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Experiment: Effect of the ribosomal protein mutation pgy1-1 on total mRNAs and on ribosome-bound mRNAs in Arabidopsis seedlings

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-656

Mutations in ribosomal proteins are known to cause specific developmental defects. For example, six different ribosomal protein mutations have been isolated as modifiers of the leaf patterning gene ASYMMETRIC LEAVES 1 (AS1). These modifiers, called piggyback (pgy), all condition ectopic outgrowths on the upper side of as1 mutant leaves (Pinon et al. 2008, Development 135, 1315).The specific developmental phenotypes of as1 pgy mutants suggested that ribosomes serve as a control point in expression of development genes. It is possible that different subpopulations of ribosomes, containing different ribosomal protein isoforms, are preferentially associated with translation or the translational control of subsets of mRNAs with specific roles in development.To test this hypothesis, we compared mRNAs pulled down with immunoprecipitated ribosomes in the pgy1-1 mutant and in the PGY1 control. To tag ribosomes for immunoprecipitation, we used pgy3-1 mutant plants complemented with a functional PGY3-FLAG fusion. As controls for changes in steady-state mRNA levels, we also compared RNA from total homogenates from pgy1-1 pgy3-1 PGY3-FLAG and from pgy3-1 PGY3-FLAG plants. Two independent experiments were performed, each using three biological replicates per treatment, comparing RNA extracted from total homogenates and from immunoprecipitated ribosomes (see detailed methods).

About the Experimenter

Name:Dr Cristina Pignocchi
Head of Lab Name:Dr Robert Sablowski
Lab:
Address:Cell and Developmental Biology
John Innes Centre
Norwich Research Park
Postcode: NR4 7UH
Country: United Kingdom
 
Telephone Number: 441603450530

All of the data available in this website/database is free, and you are free to do whatever you please with it. If you intend to publish work based on any of this data, please acknowledge us, contact the experimenter above, and either acknowledge them or use them as co-authors in the work.

About this Experiment

Experiment Type: gene_knock_out
Number of Slides:24
 
Experimental Parameters:
parametergene_knock_out
Quality Control Measures Taken:
References:
Reference Pinon et al. 2008, Development 135, 1315
 
Other Information:

Slides in this Experiment

Hybridisation Set: Pignocchi: Effect of the ribosomal protein mutation pgy1-1 on total mRNAs and on ribosome-bound mRNAs in Arabidopsis seedlings

Slide: Pignocchi_exp2_wt_IP_rep1_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_wt_IP_rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_wt_IP_rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.456902
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.209436
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.590099
Spike_AFFX-r2-Bs-dap_5_signal134.455383
NoiseAvg:3.06,Std:0.09,Min:2.9,Max:3.4
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal48.551987
#P13776
Spike_AFFX-r2-Bs-phe_M_signal81.829346
Corner-Avg:15432,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal43.332924
Spike_AFFX-r2-Ec-bioB_3_signal24.953411
Spike_AFFX-r2-Bs-lys_M_signal49.913189
Spike_AFFX-r2-P1-cre_3_signal4301.667969
Spike_AFFX-r2-Bs-lys_3-5-ratio1.432385
Spike_AFFX-r2-Bs-dap_M_signal509.586395
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio5.827981
Spike_AFFX-r2-Ec-bioB_avg-signal36.857708
Spike_AFFX-r2-Bs-thr_avg-signal161.836609
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1150.081543
Spike_AFFX-r2-Bs-phe_5_signal99.202141
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1385.937378
RawQ2.408641
Spike_AFFX-r2-Bs-lys_5_signal71.539436
Signal(A)4.948442
%A37.321350
Signal(All)148.235733
Corner+Avg:320,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal289.294159
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal152.604675
Spike_AFFX-r2-Ec-bioD_avg-signal1268.009521
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P60.394566
Spike_AFFX-r2-Bs-lys_avg-signal74.641563
Spike_AFFX-r2-P1-cre_avg-signal3929.212402
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.284086
Spike_AFFX-r2-Bs-thr_3_signal282.960083
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal111.212059
Spike_AFFX-r2-Bs-dap_3_signal784.444702
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.205078
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal327.024994
#M521
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal102.472046
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.884624
Spike_AFFX-r2-Bs-thr_M_signal153.997757
Signal(P)241.731964
Spike_AFFX-r2-Bs-phe_3-5-ratio1.538320
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:12110,Count:9
Spike_AFFX-r2-P1-cre_5_signal3556.756592
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8513
Signal(M)17.335131
BackgroundAvg:56.01,Std:0.51,Min:55.2,Max:57.7
Spike_AFFX-r2-Ec-bioC_avg-signal308.159576
Spike_AFFX-r2-Bs-dap_avg-signal476.162201
Spike_AFFX-r2-Bs-dap_3-5-ratio5.834238
Spike_AFFX-r2-Ec-bioB_5_signal42.286793
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.456902
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_wt_TOT_rep2_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_wt_TOT_rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_wt_TOT_rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 PGY3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.841489
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.142096
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.305189
Spike_AFFX-r2-Bs-dap_5_signal18.840338
NoiseAvg:2.46,Std:0.08,Min:2.3,Max:2.7
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal8.040279
#P14100
Spike_AFFX-r2-Bs-phe_M_signal36.044006
Corner-Avg:14874,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal201.568634
Spike_AFFX-r2-Ec-bioB_3_signal184.842438
Spike_AFFX-r2-Bs-lys_M_signal23.617840
Spike_AFFX-r2-P1-cre_3_signal8626.251953
Spike_AFFX-r2-Bs-lys_3-5-ratio1.711968
Spike_AFFX-r2-Bs-dap_M_signal152.877640
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio21.706921
Spike_AFFX-r2-Ec-bioB_avg-signal176.010742
Spike_AFFX-r2-Bs-thr_avg-signal78.834969
Spike_AFFX-r2-Bs-thr_5_detectionA
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1802.368774
Spike_AFFX-r2-Bs-phe_5_signal33.690582
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2306.411377
RawQ2.378701
Spike_AFFX-r2-Bs-lys_5_signal31.826674
Signal(A)5.561879
%A35.900921
Signal(All)157.664902
Corner+Avg:233,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal604.488647
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal149.197708
Spike_AFFX-r2-Ec-bioD_avg-signal2054.390137
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.814995
Spike_AFFX-r2-Bs-lys_avg-signal36.643585
Spike_AFFX-r2-P1-cre_avg-signal8089.626953
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.284086
Spike_AFFX-r2-Bs-thr_3_signal174.529709
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal72.977432
Spike_AFFX-r2-Bs-dap_3_signal713.846497
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.279656
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal528.482971
#M521
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal54.486244
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.143819
Spike_AFFX-r2-Bs-thr_M_signal53.934929
Signal(P)251.107330
Spike_AFFX-r2-Bs-phe_3-5-ratio4.428469
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:13221,Count:9
Spike_AFFX-r2-P1-cre_5_signal7553.001465
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8189
Signal(M)19.532808
BackgroundAvg:59.12,Std:0.81,Min:57.3,Max:61.0
Spike_AFFX-r2-Ec-bioC_avg-signal566.485840
Spike_AFFX-r2-Bs-dap_avg-signal295.188141
Spike_AFFX-r2-Bs-dap_3-5-ratio37.889263
Spike_AFFX-r2-Ec-bioB_5_signal141.621155
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.841489
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_wt_IP_rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_wt_IP_rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_wt_IP_rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.687013
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.231142
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.960565
Spike_AFFX-r2-Bs-dap_5_signal174.198090
NoiseAvg:2.69,Std:0.05,Min:2.5,Max:2.9
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal66.583870
#P13522
Spike_AFFX-r2-Bs-phe_M_signal85.200302
Corner-Avg:15564,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal135.689087
Spike_AFFX-r2-Ec-bioB_3_signal102.251640
Spike_AFFX-r2-Bs-lys_M_signal29.016861
Spike_AFFX-r2-P1-cre_3_signal5800.624023
Spike_AFFX-r2-Bs-lys_3-5-ratio1.088467
Spike_AFFX-r2-Bs-dap_M_signal455.800995
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.601686
Spike_AFFX-r2-Ec-bioB_avg-signal114.796730
Spike_AFFX-r2-Bs-thr_avg-signal147.004471
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1165.078003
Spike_AFFX-r2-Bs-phe_5_signal119.318436
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1536.620972
RawQ2.610496
Spike_AFFX-r2-Bs-lys_5_signal62.665951
Signal(A)4.771955
%A38.706707
Signal(All)154.910782
Corner+Avg:252,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal344.976501
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal147.626053
Spike_AFFX-r2-Ec-bioD_avg-signal1350.849487
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P59.281017
Spike_AFFX-r2-Bs-lys_avg-signal53.297535
Spike_AFFX-r2-P1-cre_avg-signal5256.101563
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.012275
Spike_AFFX-r2-Bs-thr_3_signal239.814194
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal117.381592
Spike_AFFX-r2-Bs-dap_3_signal708.910034
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.318900
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal352.731659
#M459
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal68.209801
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.978014
Spike_AFFX-r2-Bs-thr_M_signal134.615356
Signal(P)257.623596
Spike_AFFX-r2-Bs-phe_3-5-ratio1.237244
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:11899,Count:9
Spike_AFFX-r2-P1-cre_5_signal4711.579590
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8829
Signal(M)16.987068
BackgroundAvg:61.56,Std:0.57,Min:60.0,Max:63.0
Spike_AFFX-r2-Ec-bioC_avg-signal348.854065
Spike_AFFX-r2-Bs-dap_avg-signal446.303070
Spike_AFFX-r2-Bs-dap_3-5-ratio4.069562
Spike_AFFX-r2-Ec-bioB_5_signal106.449478
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.687013
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_pgy1_TOT_rep2_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_pgy1_TOT_rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_pgy1_TOT_rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.413653
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio0.974272
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.753081
Spike_AFFX-r2-Bs-dap_5_signal17.097527
NoiseAvg:2.67,Std:0.07,Min:2.5,Max:2.9
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal9.087649
#P12646
Spike_AFFX-r2-Bs-phe_M_signal17.739967
Corner-Avg:13654,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal244.584137
Spike_AFFX-r2-Ec-bioB_3_signal194.332306
Spike_AFFX-r2-Bs-lys_M_signal25.154131
Spike_AFFX-r2-P1-cre_3_signal9551.922852
Spike_AFFX-r2-Bs-lys_3-5-ratio3.746168
Spike_AFFX-r2-Bs-dap_M_signal183.764816
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio23.844213
Spike_AFFX-r2-Ec-bioB_avg-signal183.256104
Spike_AFFX-r2-Bs-thr_avg-signal97.030327
Spike_AFFX-r2-Bs-thr_5_detectionA
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal2450.797852
Spike_AFFX-r2-Bs-phe_5_signal13.094188
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2802.393311
RawQ2.644736
Spike_AFFX-r2-Bs-lys_5_signal19.036833
Signal(A)10.746593
%A41.486191
Signal(All)160.937088
Corner+Avg:228,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal700.897095
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal107.094162
Spike_AFFX-r2-Ec-bioD_avg-signal2626.595703
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionA
%P55.440598
Spike_AFFX-r2-Bs-lys_avg-signal38.502045
Spike_AFFX-r2-P1-cre_avg-signal9678.045898
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M3.073214
Spike_AFFX-r2-Bs-thr_3_signal216.687851
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal45.976105
Spike_AFFX-r2-Bs-dap_3_signal721.074219
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.143462
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal703.993835
#M701
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal71.315170
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.995601
Spike_AFFX-r2-Bs-thr_M_signal65.315468
Signal(P)280.491455
Spike_AFFX-r2-Bs-phe_3-5-ratio8.178756
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:9801,Count:9
Spike_AFFX-r2-P1-cre_5_signal9804.168945
Spike_AFFX-r2-Bs-phe_M_detectionA
#A9463
Signal(M)31.647858
BackgroundAvg:62.96,Std:0.55,Min:61.4,Max:64.5
Spike_AFFX-r2-Ec-bioC_avg-signal702.445435
Spike_AFFX-r2-Bs-dap_avg-signal307.312195
Spike_AFFX-r2-Bs-dap_3-5-ratio42.174183
Spike_AFFX-r2-Ec-bioB_5_signal110.851860
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF1.413653
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_pgy1_IP_rep1_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_pgy1_IP_rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_pgy1_IP_rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.89644
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.212700
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.821396
Spike_AFFX-r2-Bs-dap_5_signal105.355598
NoiseAvg:2.59,Std:0.09,Min:2.4,Max:2.9
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal37.974449
#P13379
Spike_AFFX-r2-Bs-phe_M_signal112.090202
Corner-Avg:12916,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal140.383774
Spike_AFFX-r2-Ec-bioB_3_signal106.151840
Spike_AFFX-r2-Bs-lys_M_signal41.415840
Spike_AFFX-r2-P1-cre_3_signal6846.512207
Spike_AFFX-r2-Bs-lys_3-5-ratio1.151096
Spike_AFFX-r2-Bs-dap_M_signal439.958038
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio7.025573
Spike_AFFX-r2-Ec-bioB_avg-signal125.256348
Spike_AFFX-r2-Bs-thr_avg-signal150.925400
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1200.196045
Spike_AFFX-r2-Bs-phe_5_signal139.704285
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1633.069214
RawQ2.598391
Spike_AFFX-r2-Bs-lys_5_signal67.068871
Signal(A)6.313110
%A38.982903
Signal(All)160.020416
Corner+Avg:196,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal401.868378
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal165.433762
Spike_AFFX-r2-Ec-bioD_avg-signal1416.632568
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P58.654099
Spike_AFFX-r2-Bs-lys_avg-signal61.895798
Spike_AFFX-r2-P1-cre_avg-signal6246.095703
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.362999
Spike_AFFX-r2-Bs-thr_3_signal266.792267
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal139.076080
Spike_AFFX-r2-Bs-dap_3_signal763.616699
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.360669
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal390.408508
#M539
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal77.202682
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.029353
Spike_AFFX-r2-Bs-thr_M_signal148.009491
Signal(P)267.721924
Spike_AFFX-r2-Bs-phe_3-5-ratio1.184171
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:12512,Count:9
Spike_AFFX-r2-P1-cre_5_signal5645.679199
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8892
Signal(M)22.408405
BackgroundAvg:60.70,Std:0.74,Min:58.5,Max:62.4
Spike_AFFX-r2-Ec-bioC_avg-signal396.138428
Spike_AFFX-r2-Bs-dap_avg-signal436.310089
Spike_AFFX-r2-Bs-dap_3-5-ratio7.247993
Spike_AFFX-r2-Ec-bioB_5_signal129.233414
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.896440
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_wt_TOT_rep3_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_wt_TOT_rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_wt_TOT_rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 PGY3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.699538
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio0.938753
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.278951
Spike_AFFX-r2-Bs-dap_5_signal107.457428
NoiseAvg:2.47,Std:0.10,Min:2.2,Max:2.8
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal30.489328
#P14622
Spike_AFFX-r2-Bs-phe_M_signal83.636269
Corner-Avg:11352,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal47.476177
Spike_AFFX-r2-Ec-bioB_3_signal36.172050
Spike_AFFX-r2-Bs-lys_M_signal46.965290
Spike_AFFX-r2-P1-cre_3_signal4144.109863
Spike_AFFX-r2-Bs-lys_3-5-ratio1.234560
Spike_AFFX-r2-Bs-dap_M_signal469.589539
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio12.643205
Spike_AFFX-r2-Ec-bioB_avg-signal37.310268
Spike_AFFX-r2-Bs-thr_avg-signal186.238876
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1176.839233
Spike_AFFX-r2-Bs-phe_5_signal112.939545
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1454.418335
RawQ2.414338
Spike_AFFX-r2-Bs-lys_5_signal82.695808
Signal(A)5.167781
%A33.849190
Signal(All)144.714767
Corner+Avg:191,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal315.879211
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal146.637390
Spike_AFFX-r2-Ec-bioD_avg-signal1315.628784
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P64.103462
Spike_AFFX-r2-Bs-lys_avg-signal77.251335
Spike_AFFX-r2-P1-cre_avg-signal4279.295898
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.047348
Spike_AFFX-r2-Bs-thr_3_signal385.482819
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal114.404396
Spike_AFFX-r2-Bs-dap_3_signal938.111267
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.235868
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal321.152344
#M467
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal102.092911
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.983581
Spike_AFFX-r2-Bs-thr_M_signal142.744492
Signal(P)222.473740
Spike_AFFX-r2-Bs-phe_3-5-ratio1.298371
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:9307,Count:9
Spike_AFFX-r2-P1-cre_5_signal4414.481445
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7721
Signal(M)17.199781
BackgroundAvg:57.23,Std:0.60,Min:55.8,Max:59.3
Spike_AFFX-r2-Ec-bioC_avg-signal318.515778
Spike_AFFX-r2-Bs-dap_avg-signal505.052734
Spike_AFFX-r2-Bs-dap_3-5-ratio8.730074
Spike_AFFX-r2-Ec-bioB_5_signal28.282587
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.699538
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_wt_TOT_rep2_ATH

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_wt_TOT_rep2_ATH") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_wt_TOT_rep2_ATH
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 PGY3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.722451
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.194712
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.677956
Spike_AFFX-r2-Bs-dap_5_signal116.628113
NoiseAvg:2.62,Std:0.06,Min:2.5,Max:2.8
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal35.066242
#P14309
Spike_AFFX-r2-Bs-phe_M_signal97.593231
Corner-Avg:11934,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal46.122169
Spike_AFFX-r2-Ec-bioB_3_signal26.920399
Spike_AFFX-r2-Bs-lys_M_signal62.127598
Spike_AFFX-r2-P1-cre_3_signal5721.507324
Spike_AFFX-r2-Bs-lys_3-5-ratio1.514718
Spike_AFFX-r2-Bs-dap_M_signal519.036194
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio14.034892
Spike_AFFX-r2-Ec-bioB_avg-signal37.583591
Spike_AFFX-r2-Bs-thr_avg-signal229.260513
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1369.114380
Spike_AFFX-r2-Bs-phe_5_signal123.911270
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1737.428833
RawQ2.369270
Spike_AFFX-r2-Bs-lys_5_signal86.980911
Signal(A)5.661501
%A35.168785
Signal(All)149.791046
Corner+Avg:208,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal310.953430
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal171.673813
Spike_AFFX-r2-Ec-bioD_avg-signal1553.271606
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P62.731258
Spike_AFFX-r2-Bs-lys_avg-signal93.620033
Spike_AFFX-r2-P1-cre_avg-signal5255.266602
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.099956
Spike_AFFX-r2-Bs-thr_3_signal492.150909
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal131.059433
Spike_AFFX-r2-Bs-dap_3_signal1038.940186
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.269017
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal333.010101
#M479
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal131.751587
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.933766
Spike_AFFX-r2-Bs-thr_M_signal160.564453
Signal(P)234.933517
Spike_AFFX-r2-Bs-phe_3-5-ratio1.385458
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:8493,Count:9
Spike_AFFX-r2-P1-cre_5_signal4789.025391
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8022
Signal(M)20.153187
BackgroundAvg:57.66,Std:0.25,Min:57.1,Max:58.6
Spike_AFFX-r2-Ec-bioC_avg-signal321.981750
Spike_AFFX-r2-Bs-dap_avg-signal558.201477
Spike_AFFX-r2-Bs-dap_3-5-ratio8.908145
Spike_AFFX-r2-Ec-bioB_5_signal39.708199
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.722451
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_wt_TOT_rep1_ATH

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_wt_TOT_rep1_ATH") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_wt_TOT_rep1_ATH
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 PGY3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.733706
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.272933
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.050161
Spike_AFFX-r2-Bs-dap_5_signal89.644478
NoiseAvg:2.66,Std:0.06,Min:2.4,Max:2.8
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal16.457834
#P13854
Spike_AFFX-r2-Bs-phe_M_signal89.572838
Corner-Avg:12817,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal40.006786
Spike_AFFX-r2-Ec-bioB_3_signal32.639824
Spike_AFFX-r2-Bs-lys_M_signal51.882591
Spike_AFFX-r2-P1-cre_3_signal6177.375488
Spike_AFFX-r2-Bs-lys_3-5-ratio1.471257
Spike_AFFX-r2-Bs-dap_M_signal495.834717
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio21.639681
Spike_AFFX-r2-Ec-bioB_avg-signal34.575798
Spike_AFFX-r2-Bs-thr_avg-signal162.608566
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1453.183594
Spike_AFFX-r2-Bs-phe_5_signal76.404915
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1763.794678
RawQ2.390263
Spike_AFFX-r2-Bs-lys_5_signal75.196709
Signal(A)6.235955
%A36.957474
Signal(All)152.159256
Corner+Avg:210,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal308.730621
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal182.018402
Spike_AFFX-r2-Ec-bioD_avg-signal1608.489136
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P60.736519
Spike_AFFX-r2-Bs-lys_avg-signal79.237671
Spike_AFFX-r2-P1-cre_avg-signal5515.121094
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.306006
Spike_AFFX-r2-Bs-thr_3_signal356.142273
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal115.998718
Spike_AFFX-r2-Bs-dap_3_signal851.752441
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.213745
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal328.955994
#M526
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal110.633720
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.938516
Spike_AFFX-r2-Bs-thr_M_signal115.225624
Signal(P)245.918884
Spike_AFFX-r2-Bs-phe_3-5-ratio2.382287
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10280,Count:9
Spike_AFFX-r2-P1-cre_5_signal4852.866699
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8430
Signal(M)21.336897
BackgroundAvg:57.37,Std:0.54,Min:55.7,Max:58.6
Spike_AFFX-r2-Ec-bioC_avg-signal318.843323
Spike_AFFX-r2-Bs-dap_avg-signal479.077240
Spike_AFFX-r2-Bs-dap_3-5-ratio9.501449
Spike_AFFX-r2-Ec-bioB_5_signal31.080790
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.733706
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_wt_IP_rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_wt_IP_rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_wt_IP_rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.521447
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.236274
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.961581
Spike_AFFX-r2-Bs-dap_5_signal132.863190
NoiseAvg:3.09,Std:0.08,Min:2.8,Max:3.3
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal43.299767
#P14040
Spike_AFFX-r2-Bs-phe_M_signal90.134857
Corner-Avg:11422,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal32.792587
Spike_AFFX-r2-Ec-bioB_3_signal25.669746
Spike_AFFX-r2-Bs-lys_M_signal39.753609
Spike_AFFX-r2-P1-cre_3_signal4176.097656
Spike_AFFX-r2-Bs-lys_3-5-ratio1.336443
Spike_AFFX-r2-Bs-dap_M_signal459.233582
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio8.499039
Spike_AFFX-r2-Ec-bioB_avg-signal28.385900
Spike_AFFX-r2-Bs-thr_avg-signal191.187332
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal906.887268
Spike_AFFX-r2-Bs-phe_5_signal99.858063
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1234.486450
RawQ2.667857
Spike_AFFX-r2-Bs-lys_5_signal66.009499
Signal(A)5.318243
%A35.918457
Signal(All)145.404724
Corner+Avg:200,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal298.276123
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal131.308640
Spike_AFFX-r2-Ec-bioD_avg-signal1070.686890
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.551952
Spike_AFFX-r2-Bs-lys_avg-signal64.660347
Spike_AFFX-r2-P1-cre_avg-signal3777.034180
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.529592
Spike_AFFX-r2-Bs-thr_3_signal368.006409
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal107.100525
Spike_AFFX-r2-Bs-dap_3_signal752.742249
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.361235
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal301.686615
#M577
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal88.217941
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.988695
Spike_AFFX-r2-Bs-thr_M_signal162.255859
Signal(P)232.419418
Spike_AFFX-r2-Bs-phe_3-5-ratio1.314953
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10201,Count:9
Spike_AFFX-r2-P1-cre_5_signal3377.970459
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8193
Signal(M)17.228527
BackgroundAvg:63.01,Std:0.52,Min:61.3,Max:64.3
Spike_AFFX-r2-Ec-bioC_avg-signal299.981384
Spike_AFFX-r2-Bs-dap_avg-signal448.279663
Spike_AFFX-r2-Bs-dap_3-5-ratio5.665544
Spike_AFFX-r2-Ec-bioB_5_signal26.695362
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.521447
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_wt_IP_rep2_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_wt_IP_rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_wt_IP_rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.528753
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.231430
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.677126
Spike_AFFX-r2-Bs-dap_5_signal87.336746
NoiseAvg:3.30,Std:0.10,Min:3.0,Max:3.6
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal36.391769
#P13797
Spike_AFFX-r2-Bs-phe_M_signal63.463577
Corner-Avg:11249,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal35.099781
Spike_AFFX-r2-Ec-bioB_3_signal26.145252
Spike_AFFX-r2-Bs-lys_M_signal37.589451
Spike_AFFX-r2-P1-cre_3_signal4126.095215
Spike_AFFX-r2-Bs-lys_3-5-ratio1.402144
Spike_AFFX-r2-Bs-dap_M_signal370.754486
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio9.098468
Spike_AFFX-r2-Ec-bioB_avg-signal33.285706
Spike_AFFX-r2-Bs-thr_avg-signal163.015808
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal858.523499
Spike_AFFX-r2-Bs-phe_5_signal72.172295
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1177.367432
RawQ2.786691
Spike_AFFX-r2-Bs-lys_5_signal57.851631
Signal(A)5.632206
%A36.904865
Signal(All)146.765961
Corner+Avg:209,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal291.647186
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal128.363846
Spike_AFFX-r2-Ec-bioD_avg-signal1017.945435
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P60.486629
Spike_AFFX-r2-Bs-lys_avg-signal58.852467
Spike_AFFX-r2-P1-cre_avg-signal3738.374023
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.608505
Spike_AFFX-r2-Bs-thr_3_signal331.109344
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal87.999908
Spike_AFFX-r2-Bs-dap_3_signal686.596802
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.371386
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal293.554291
#M595
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal81.116325
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.993503
Spike_AFFX-r2-Bs-thr_M_signal121.546295
Signal(P)238.411407
Spike_AFFX-r2-Bs-phe_3-5-ratio1.778575
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:9561,Count:9
Spike_AFFX-r2-P1-cre_5_signal3350.653076
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8418
Signal(M)18.415613
BackgroundAvg:66.79,Std:0.87,Min:65.1,Max:69.6
Spike_AFFX-r2-Ec-bioC_avg-signal292.600739
Spike_AFFX-r2-Bs-dap_avg-signal381.562714
Spike_AFFX-r2-Bs-dap_3-5-ratio7.861488
Spike_AFFX-r2-Ec-bioB_5_signal38.612080
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.528753
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_pgy1_IP_rep3_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_pgy1_IP_rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_pgy1_IP_rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.864906
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.186076
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.203705
Spike_AFFX-r2-Bs-dap_5_signal284.064545
NoiseAvg:2.57,Std:0.05,Min:2.4,Max:2.8
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal112.787315
#P13580
Spike_AFFX-r2-Bs-phe_M_signal143.113266
Corner-Avg:14483,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal220.883713
Spike_AFFX-r2-Ec-bioB_3_signal149.770355
Spike_AFFX-r2-Bs-lys_M_signal45.654156
Spike_AFFX-r2-P1-cre_3_signal8014.083496
Spike_AFFX-r2-Bs-lys_3-5-ratio1.084134
Spike_AFFX-r2-Bs-dap_M_signal723.676819
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.411849
Spike_AFFX-r2-Ec-bioB_avg-signal165.026169
Spike_AFFX-r2-Bs-thr_avg-signal257.018097
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1702.988892
Spike_AFFX-r2-Bs-phe_5_signal211.259750
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2222.274902
RawQ2.585240
Spike_AFFX-r2-Bs-lys_5_signal88.934212
Signal(A)5.799971
%A37.913197
Signal(All)162.128662
Corner+Avg:228,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal581.651245
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal212.545227
Spike_AFFX-r2-Ec-bioD_avg-signal1962.631836
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P59.535290
Spike_AFFX-r2-Bs-lys_avg-signal77.001671
Spike_AFFX-r2-P1-cre_avg-signal7385.443359
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.551512
Spike_AFFX-r2-Bs-thr_3_signal384.813232
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal188.972763
Spike_AFFX-r2-Bs-dap_3_signal1096.500732
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.304926
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal555.841736
#M582
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal96.416649
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.046433
Spike_AFFX-r2-Bs-thr_M_signal273.453705
Signal(P)267.732910
Spike_AFFX-r2-Bs-phe_3-5-ratio1.006085
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:11672,Count:9
Spike_AFFX-r2-P1-cre_5_signal6756.802734
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8648
Signal(M)20.934410
BackgroundAvg:59.86,Std:0.33,Min:59.0,Max:61.0
Spike_AFFX-r2-Ec-bioC_avg-signal568.746460
Spike_AFFX-r2-Bs-dap_avg-signal701.414001
Spike_AFFX-r2-Bs-dap_3-5-ratio3.860041
Spike_AFFX-r2-Ec-bioB_5_signal124.424477
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.864906
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_pgy1_IP_rep2_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_pgy1_IP_rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_pgy1_IP_rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.901426
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.087589
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.867870
Spike_AFFX-r2-Bs-dap_5_signal170.847229
NoiseAvg:2.63,Std:0.06,Min:2.4,Max:2.8
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal74.914665
#P13317
Spike_AFFX-r2-Bs-phe_M_signal106.399422
Corner-Avg:14727,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal156.990952
Spike_AFFX-r2-Ec-bioB_3_signal94.179405
Spike_AFFX-r2-Bs-lys_M_signal26.776955
Spike_AFFX-r2-P1-cre_3_signal5993.012695
Spike_AFFX-r2-Bs-lys_3-5-ratio1.560107
Spike_AFFX-r2-Bs-dap_M_signal416.992188
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio4.151595
Spike_AFFX-r2-Ec-bioB_avg-signal119.896080
Spike_AFFX-r2-Bs-thr_avg-signal180.225952
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1305.839966
Spike_AFFX-r2-Bs-phe_5_signal126.026878
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1830.450684
RawQ2.700693
Spike_AFFX-r2-Bs-lys_5_signal49.300747
Signal(A)6.227457
%A39.048664
Signal(All)159.958893
Corner+Avg:236,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal359.898346
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal130.816116
Spike_AFFX-r2-Ec-bioD_avg-signal1568.145264
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P58.382290
Spike_AFFX-r2-Bs-lys_avg-signal50.997379
Spike_AFFX-r2-P1-cre_avg-signal5751.689941
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.569049
Spike_AFFX-r2-Bs-thr_3_signal311.015320
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal121.080811
Spike_AFFX-r2-Bs-dap_3_signal720.861328
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.401742
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal391.631439
#M586
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal76.914436
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.918972
Spike_AFFX-r2-Bs-thr_M_signal154.747849
Signal(P)268.843353
Spike_AFFX-r2-Bs-phe_3-5-ratio1.038002
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10609,Count:9
Spike_AFFX-r2-P1-cre_5_signal5510.367188
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8907
Signal(M)22.197208
BackgroundAvg:62.01,Std:0.29,Min:60.7,Max:63.0
Spike_AFFX-r2-Ec-bioC_avg-signal375.764893
Spike_AFFX-r2-Bs-dap_avg-signal436.233551
Spike_AFFX-r2-Bs-dap_3-5-ratio4.219333
Spike_AFFX-r2-Ec-bioB_5_signal108.517876
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.901426
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_wt_IP_rep1_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_wt_IP_rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_wt_IP_rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.579649
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.274434
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.226489
Spike_AFFX-r2-Bs-dap_5_signal46.174519
NoiseAvg:3.08,Std:0.07,Min:2.9,Max:3.3
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal16.707081
#P13595
Spike_AFFX-r2-Bs-phe_M_signal56.556759
Corner-Avg:14528,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal80.545471
Spike_AFFX-r2-Ec-bioB_3_signal75.707069
Spike_AFFX-r2-Bs-lys_M_signal18.419434
Spike_AFFX-r2-P1-cre_3_signal4001.103271
Spike_AFFX-r2-Bs-lys_3-5-ratio1.723822
Spike_AFFX-r2-Bs-dap_M_signal185.828369
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio6.215153
Spike_AFFX-r2-Ec-bioB_avg-signal72.659729
Spike_AFFX-r2-Bs-thr_avg-signal60.539215
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal642.693481
Spike_AFFX-r2-Bs-phe_5_signal69.921303
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal902.644165
RawQ2.790374
Spike_AFFX-r2-Bs-lys_5_signal33.755764
Signal(A)5.499019
%A38.022797
Signal(All)151.940292
Corner+Avg:235,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal240.574463
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal82.044731
Spike_AFFX-r2-Ec-bioD_avg-signal772.668823
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P59.601051
Spike_AFFX-r2-Bs-lys_avg-signal36.788040
Spike_AFFX-r2-P1-cre_avg-signal3570.308838
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.376151
Spike_AFFX-r2-Bs-thr_3_signal103.837067
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal69.507599
Spike_AFFX-r2-Bs-dap_3_signal328.847321
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.404471
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal247.684799
#M542
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal58.188923
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.971293
Spike_AFFX-r2-Bs-thr_M_signal61.073498
Signal(P)250.713089
Spike_AFFX-r2-Bs-phe_3-5-ratio1.173387
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:12876,Count:9
Spike_AFFX-r2-P1-cre_5_signal3139.514404
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8673
Signal(M)17.750542
BackgroundAvg:63.67,Std:0.67,Min:62.0,Max:65.1
Spike_AFFX-r2-Ec-bioC_avg-signal244.129639
Spike_AFFX-r2-Bs-dap_avg-signal186.950058
Spike_AFFX-r2-Bs-dap_3-5-ratio7.121835
Spike_AFFX-r2-Ec-bioB_5_signal61.726646
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.579649
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_pgy1_TOT_rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_pgy1_TOT_rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_pgy1_TOT_rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.90464
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.173964
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.992688
Spike_AFFX-r2-Bs-dap_5_signal26.165636
NoiseAvg:2.83,Std:0.08,Min:2.6,Max:3.1
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal6.736694
#P13854
Spike_AFFX-r2-Bs-phe_M_signal56.738167
Corner-Avg:14885,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal198.730408
Spike_AFFX-r2-Ec-bioB_3_signal145.000778
Spike_AFFX-r2-Bs-lys_M_signal38.339836
Spike_AFFX-r2-P1-cre_3_signal7710.846191
Spike_AFFX-r2-Bs-lys_3-5-ratio2.210358
Spike_AFFX-r2-Bs-dap_M_signal158.850754
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio33.101910
Spike_AFFX-r2-Ec-bioB_avg-signal163.266663
Spike_AFFX-r2-Bs-thr_avg-signal96.744240
Spike_AFFX-r2-Bs-thr_5_detectionA
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1701.622192
Spike_AFFX-r2-Bs-phe_5_signal82.028870
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2285.880859
RawQ2.737223
Spike_AFFX-r2-Bs-lys_5_signal27.601696
Signal(A)7.643464
%A36.619904
Signal(All)155.437180
Corner+Avg:239,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal538.914673
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal171.483063
Spike_AFFX-r2-Ec-bioD_avg-signal1993.751465
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P60.736519
Spike_AFFX-r2-Bs-lys_avg-signal42.317051
Spike_AFFX-r2-P1-cre_avg-signal7139.530273
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.643577
Spike_AFFX-r2-Bs-thr_3_signal222.997437
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal103.416695
Spike_AFFX-r2-Bs-dap_3_signal613.968384
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.343354
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal529.730835
#M603
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal61.009624
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.017337
Spike_AFFX-r2-Bs-thr_M_signal60.498585
Signal(P)250.251236
Spike_AFFX-r2-Bs-phe_3-5-ratio2.090521
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:12491,Count:9
Spike_AFFX-r2-P1-cre_5_signal6568.213867
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8353
Signal(M)24.370863
BackgroundAvg:66.99,Std:0.52,Min:65.1,Max:68.5
Spike_AFFX-r2-Ec-bioC_avg-signal534.322754
Spike_AFFX-r2-Bs-dap_avg-signal266.328278
Spike_AFFX-r2-Bs-dap_3-5-ratio23.464684
Spike_AFFX-r2-Ec-bioB_5_signal146.068787
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.904640
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_pgy1_TOT_rep1_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_pgy1_TOT_rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_pgy1_TOT_rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:1.424787
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.246258
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.275568
Spike_AFFX-r2-Bs-dap_5_signal13.229635
NoiseAvg:2.41,Std:0.08,Min:2.2,Max:2.6
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal15.375336
#P12719
Spike_AFFX-r2-Bs-phe_M_signal23.095919
Corner-Avg:13521,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal337.120880
Spike_AFFX-r2-Ec-bioB_3_signal296.647003
Spike_AFFX-r2-Bs-lys_M_signal22.661274
Spike_AFFX-r2-P1-cre_3_signal13323.907227
Spike_AFFX-r2-Bs-lys_3-5-ratio6.036155
Spike_AFFX-r2-Bs-dap_M_signal217.035004
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio13.733596
Spike_AFFX-r2-Ec-bioB_avg-signal288.776184
Spike_AFFX-r2-Bs-thr_avg-signal96.118660
Spike_AFFX-r2-Bs-thr_5_detectionA
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal2967.275879
Spike_AFFX-r2-Bs-phe_5_signal19.412405
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal3568.493896
RawQ2.509180
Spike_AFFX-r2-Bs-lys_5_signal11.180112
Signal(A)10.317343
%A41.266987
Signal(All)164.737473
Corner+Avg:217,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal1040.683838
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal197.395920
Spike_AFFX-r2-Ec-bioD_avg-signal3267.884766
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P55.760632
Spike_AFFX-r2-Bs-lys_avg-signal33.775425
Spike_AFFX-r2-P1-cre_avg-signal12007.521484
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionA
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.972381
Spike_AFFX-r2-Bs-thr_3_signal211.158646
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal79.968079
Spike_AFFX-r2-Bs-dap_3_signal964.879761
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.202616
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal947.944824
#M678
Spike_AFFX-r2-Bs-lys_5_detectionM
Spike_AFFX-r2-Bs-lys_3_signal67.484886
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.097832
Spike_AFFX-r2-Bs-thr_M_signal61.821995
Signal(P)286.128662
Spike_AFFX-r2-Bs-phe_3-5-ratio10.168546
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:11345,Count:9
Spike_AFFX-r2-P1-cre_5_signal10691.134766
Spike_AFFX-r2-Bs-phe_M_detectionM
#A9413
Signal(M)31.378119
BackgroundAvg:58.99,Std:0.63,Min:57.6,Max:60.4
Spike_AFFX-r2-Ec-bioC_avg-signal994.314331
Spike_AFFX-r2-Bs-dap_avg-signal398.381470
Spike_AFFX-r2-Bs-dap_3-5-ratio72.933212
Spike_AFFX-r2-Ec-bioB_5_signal232.560684
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF1.424787
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_wt_TOT_rep1_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_wt_TOT_rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_wt_TOT_rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 PGY3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.932486
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.195653
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.711524
Spike_AFFX-r2-Bs-dap_5_signal29.420938
NoiseAvg:2.75,Std:0.07,Min:2.6,Max:3.0
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal11.220428
#P13211
Spike_AFFX-r2-Bs-phe_M_signal37.895916
Corner-Avg:16102,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal228.465607
Spike_AFFX-r2-Ec-bioB_3_signal176.389267
Spike_AFFX-r2-Bs-lys_M_signal20.145958
Spike_AFFX-r2-P1-cre_3_signal9558.379883
Spike_AFFX-r2-Bs-lys_3-5-ratio1.740177
Spike_AFFX-r2-Bs-dap_M_signal170.300827
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio18.333559
Spike_AFFX-r2-Ec-bioB_avg-signal169.304886
Spike_AFFX-r2-Bs-thr_avg-signal86.507423
Spike_AFFX-r2-Bs-thr_5_detectionA
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal2149.182861
Spike_AFFX-r2-Bs-phe_5_signal23.037891
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2734.452148
RawQ2.741989
Spike_AFFX-r2-Bs-lys_5_signal39.650249
Signal(A)7.398304
%A39.302937
Signal(All)165.190674
Corner+Avg:250,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal631.899231
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal171.232895
Spike_AFFX-r2-Ec-bioD_avg-signal2441.817383
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P57.917580
Spike_AFFX-r2-Bs-lys_avg-signal42.931553
Spike_AFFX-r2-P1-cre_avg-signal8776.327148
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.779483
Spike_AFFX-r2-Bs-thr_3_signal205.710373
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal77.388901
Spike_AFFX-r2-Bs-dap_3_signal703.843689
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.272322
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal599.266785
#M634
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal68.998451
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.054454
Spike_AFFX-r2-Bs-thr_M_signal42.591476
Signal(P)279.077209
Spike_AFFX-r2-Bs-phe_3-5-ratio7.432663
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:13643,Count:9
Spike_AFFX-r2-P1-cre_5_signal7994.274414
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8965
Signal(M)23.319363
BackgroundAvg:63.53,Std:0.64,Min:61.4,Max:65.4
Spike_AFFX-r2-Ec-bioC_avg-signal615.583008
Spike_AFFX-r2-Bs-dap_avg-signal301.188507
Spike_AFFX-r2-Bs-dap_3-5-ratio23.923223
Spike_AFFX-r2-Ec-bioB_5_signal103.059799
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.932486
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_wt_IP_rep2_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_wt_IP_rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_wt_IP_rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.496833
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.086599
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.870354
Spike_AFFX-r2-Bs-dap_5_signal83.281906
NoiseAvg:3.15,Std:0.05,Min:3.0,Max:3.4
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal37.346081
#P14569
Spike_AFFX-r2-Bs-phe_M_signal59.961266
Corner-Avg:14397,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal93.911789
Spike_AFFX-r2-Ec-bioB_3_signal56.846184
Spike_AFFX-r2-Bs-lys_M_signal20.578329
Spike_AFFX-r2-P1-cre_3_signal3679.856934
Spike_AFFX-r2-Bs-lys_3-5-ratio1.358358
Spike_AFFX-r2-Bs-dap_M_signal221.058746
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio3.036256
Spike_AFFX-r2-Ec-bioB_avg-signal72.023933
Spike_AFFX-r2-Bs-thr_avg-signal75.390526
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal732.404785
Spike_AFFX-r2-Bs-phe_5_signal84.653229
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal983.077881
RawQ2.815686
Spike_AFFX-r2-Bs-lys_5_signal30.929136
Signal(A)4.002200
%A34.287594
Signal(All)145.584808
Corner+Avg:237,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal209.153946
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal75.093315
Spike_AFFX-r2-Ec-bioD_avg-signal857.741333
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.871109
Spike_AFFX-r2-Bs-lys_avg-signal31.173433
Spike_AFFX-r2-P1-cre_avg-signal3533.219727
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M1.841298
Spike_AFFX-r2-Bs-thr_3_signal113.392265
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal73.235939
Spike_AFFX-r2-Bs-dap_3_signal361.101227
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.342260
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal220.802734
#M420
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal42.012833
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.947243
Spike_AFFX-r2-Bs-thr_M_signal75.433220
Signal(P)225.361069
Spike_AFFX-r2-Bs-phe_3-5-ratio0.887070
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10712,Count:9
Spike_AFFX-r2-P1-cre_5_signal3386.582275
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7821
Signal(M)14.768588
BackgroundAvg:66.05,Std:0.92,Min:63.4,Max:68.0
Spike_AFFX-r2-Ec-bioC_avg-signal214.978333
Spike_AFFX-r2-Bs-dap_avg-signal221.813965
Spike_AFFX-r2-Bs-dap_3-5-ratio4.335890
Spike_AFFX-r2-Ec-bioB_5_signal65.313835
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.496833
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_pgy1_TOT_rep3_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_pgy1_TOT_rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_pgy1_TOT_rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.57151
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.208612
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.147596
Spike_AFFX-r2-Bs-dap_5_signal61.431023
NoiseAvg:3.02,Std:0.10,Min:2.8,Max:3.4
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal17.868202
#P14550
Spike_AFFX-r2-Bs-phe_M_signal71.745895
Corner-Avg:13126,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal38.799534
Spike_AFFX-r2-Ec-bioB_3_signal39.514797
Spike_AFFX-r2-Bs-lys_M_signal61.106468
Spike_AFFX-r2-P1-cre_3_signal4872.943359
Spike_AFFX-r2-Bs-lys_3-5-ratio1.003131
Spike_AFFX-r2-Bs-dap_M_signal364.406525
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio17.426512
Spike_AFFX-r2-Ec-bioB_avg-signal37.582333
Spike_AFFX-r2-Bs-thr_avg-signal138.016281
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1225.237061
Spike_AFFX-r2-Bs-phe_5_signal99.981941
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1564.135376
RawQ2.686144
Spike_AFFX-r2-Bs-lys_5_signal81.481705
Signal(A)5.279070
%A33.910564
Signal(All)145.226608
Corner+Avg:224,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal330.650085
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal143.972595
Spike_AFFX-r2-Ec-bioD_avg-signal1394.686279
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.787811
Spike_AFFX-r2-Bs-lys_avg-signal74.775009
Spike_AFFX-r2-P1-cre_avg-signal4452.396484
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.301622
Spike_AFFX-r2-Bs-thr_3_signal311.380432
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal105.233482
Spike_AFFX-r2-Bs-dap_3_signal594.845581
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.276598
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal339.424530
#M525
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal81.736847
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.974149
Spike_AFFX-r2-Bs-thr_M_signal84.800247
Signal(P)224.216949
Spike_AFFX-r2-Bs-phe_3-5-ratio1.439986
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10642,Count:9
Spike_AFFX-r2-P1-cre_5_signal4031.849609
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7735
Signal(M)17.959187
BackgroundAvg:63.94,Std:0.81,Min:62.1,Max:65.6
Spike_AFFX-r2-Ec-bioC_avg-signal335.037292
Spike_AFFX-r2-Bs-dap_avg-signal340.227692
Spike_AFFX-r2-Bs-dap_3-5-ratio9.683146
Spike_AFFX-r2-Ec-bioB_5_signal34.432667
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.571510
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_pgy1_TOT_rep2_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_pgy1_TOT_rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_pgy1_TOT_rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.463865
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.206861
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.818402
Spike_AFFX-r2-Bs-dap_5_signal46.070492
NoiseAvg:3.10,Std:0.07,Min:2.9,Max:3.3
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal16.113304
#P15065
Spike_AFFX-r2-Bs-phe_M_signal68.929947
Corner-Avg:13505,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal38.283234
Spike_AFFX-r2-Ec-bioB_3_signal33.298527
Spike_AFFX-r2-Bs-lys_M_signal44.508362
Spike_AFFX-r2-P1-cre_3_signal4224.270508
Spike_AFFX-r2-Bs-lys_3-5-ratio1.765752
Spike_AFFX-r2-Bs-dap_M_signal322.180328
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio22.742785
Spike_AFFX-r2-Ec-bioB_avg-signal37.423004
Spike_AFFX-r2-Bs-thr_avg-signal155.600861
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal937.655884
Spike_AFFX-r2-Bs-phe_5_signal67.382507
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1298.937378
RawQ2.714382
Spike_AFFX-r2-Bs-lys_5_signal64.056099
Signal(A)4.350542
%A31.937746
Signal(All)141.643799
Corner+Avg:248,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal273.577301
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal119.007080
Spike_AFFX-r2-Ec-bioD_avg-signal1118.296631
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P66.045593
Spike_AFFX-r2-Bs-lys_avg-signal73.890541
Spike_AFFX-r2-P1-cre_avg-signal3862.241943
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.016659
Spike_AFFX-r2-Bs-thr_3_signal366.461395
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal85.106514
Spike_AFFX-r2-Bs-dap_3_signal687.094849
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.385303
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal298.916138
#M460
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal113.107162
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.915231
Spike_AFFX-r2-Bs-thr_M_signal84.227875
Signal(P)211.851974
Spike_AFFX-r2-Bs-phe_3-5-ratio1.766142
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:11220,Count:9
Spike_AFFX-r2-P1-cre_5_signal3500.213379
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7285
Signal(M)16.633125
BackgroundAvg:68.15,Std:0.59,Min:66.6,Max:69.6
Spike_AFFX-r2-Ec-bioC_avg-signal286.246704
Spike_AFFX-r2-Bs-dap_avg-signal351.781860
Spike_AFFX-r2-Bs-dap_3-5-ratio14.913990
Spike_AFFX-r2-Ec-bioB_5_signal40.687256
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.463865
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_pgy1_TOT_rep1_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_pgy1_TOT_rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_pgy1_TOT_rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.510341
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.183456
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.937976
Spike_AFFX-r2-Bs-dap_5_signal71.687653
NoiseAvg:2.89,Std:0.06,Min:2.7,Max:3.1
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal21.790596
#P15044
Spike_AFFX-r2-Bs-phe_M_signal74.515121
Corner-Avg:12988,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal39.105091
Spike_AFFX-r2-Ec-bioB_3_signal25.512953
Spike_AFFX-r2-Bs-lys_M_signal57.351284
Spike_AFFX-r2-P1-cre_3_signal4870.178711
Spike_AFFX-r2-Bs-lys_3-5-ratio1.359715
Spike_AFFX-r2-Bs-dap_M_signal394.450500
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio18.418558
Spike_AFFX-r2-Ec-bioB_avg-signal30.606012
Spike_AFFX-r2-Bs-thr_avg-signal172.357742
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1189.450928
Spike_AFFX-r2-Bs-phe_5_signal77.625412
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1403.617798
RawQ2.497729
Spike_AFFX-r2-Bs-lys_5_signal70.798203
Signal(A)4.475936
%A31.920210
Signal(All)144.766174
Corner+Avg:256,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal274.383575
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal163.634079
Spike_AFFX-r2-Ec-bioD_avg-signal1296.534424
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P65.953529
Spike_AFFX-r2-Bs-lys_avg-signal74.804970
Spike_AFFX-r2-P1-cre_avg-signal4492.697266
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.126261
Spike_AFFX-r2-Bs-thr_3_signal401.351379
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal105.258202
Spike_AFFX-r2-Bs-dap_3_signal779.658936
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.180055
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal319.210938
#M485
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal96.265411
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.859568
Spike_AFFX-r2-Bs-thr_M_signal93.931252
Signal(P)216.800308
Spike_AFFX-r2-Bs-phe_3-5-ratio2.107996
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10348,Count:9
Spike_AFFX-r2-P1-cre_5_signal4115.215820
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7281
Signal(M)16.460281
BackgroundAvg:61.72,Std:0.54,Min:60.5,Max:63.0
Spike_AFFX-r2-Ec-bioC_avg-signal296.797241
Spike_AFFX-r2-Bs-dap_avg-signal415.265656
Spike_AFFX-r2-Bs-dap_3-5-ratio10.875777
Spike_AFFX-r2-Ec-bioB_5_signal27.199999
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.510341
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_pgy1_IP_rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_pgy1_IP_rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_pgy1_IP_rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.530472
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.149580
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.570559
Spike_AFFX-r2-Bs-dap_5_signal97.909470
NoiseAvg:2.78,Std:0.10,Min:2.6,Max:3.2
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal37.335442
#P14181
Spike_AFFX-r2-Bs-phe_M_signal63.973049
Corner-Avg:12486,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal37.559753
Spike_AFFX-r2-Ec-bioB_3_signal23.520409
Spike_AFFX-r2-Bs-lys_M_signal42.850597
Spike_AFFX-r2-P1-cre_3_signal4789.685059
Spike_AFFX-r2-Bs-lys_3-5-ratio1.418389
Spike_AFFX-r2-Bs-dap_M_signal367.190552
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio7.242902
Spike_AFFX-r2-Ec-bioB_avg-signal34.101208
Spike_AFFX-r2-Bs-thr_avg-signal141.037277
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1074.648438
Spike_AFFX-r2-Bs-phe_5_signal79.638062
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1388.509155
RawQ2.449154
Spike_AFFX-r2-Bs-lys_5_signal66.085686
Signal(A)4.634444
%A35.589653
Signal(All)152.454742
Corner+Avg:212,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal242.061661
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal140.462128
Spike_AFFX-r2-Ec-bioD_avg-signal1231.578857
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P62.170101
Spike_AFFX-r2-Bs-lys_avg-signal67.557159
Spike_AFFX-r2-P1-cre_avg-signal4478.074707
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.240246
Spike_AFFX-r2-Bs-thr_3_signal270.416962
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal94.691078
Spike_AFFX-r2-Bs-dap_3_signal810.822144
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.292059
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal273.567139
#M511
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal93.735191
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.884835
Spike_AFFX-r2-Bs-thr_M_signal115.359398
Signal(P)241.987244
Spike_AFFX-r2-Bs-phe_3-5-ratio1.763756
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:9932,Count:9
Spike_AFFX-r2-P1-cre_5_signal4166.464355
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8118
Signal(M)16.142918
BackgroundAvg:59.48,Std:0.43,Min:58.2,Max:60.5
Spike_AFFX-r2-Ec-bioC_avg-signal257.814392
Spike_AFFX-r2-Bs-dap_avg-signal425.307373
Spike_AFFX-r2-Bs-dap_3-5-ratio8.281345
Spike_AFFX-r2-Ec-bioB_5_signal41.223465
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.530472
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_pgy1_IP_rep2_ATH1

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_pgy1_IP_rep2_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_pgy1_IP_rep2_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.530926
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.248510
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.922986
Spike_AFFX-r2-Bs-dap_5_signal80.588402
NoiseAvg:2.76,Std:0.07,Min:2.6,Max:3.0
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal24.329659
#P14472
Spike_AFFX-r2-Bs-phe_M_signal59.236019
Corner-Avg:11271,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal34.544903
Spike_AFFX-r2-Ec-bioB_3_signal27.642647
Spike_AFFX-r2-Bs-lys_M_signal46.806801
Spike_AFFX-r2-P1-cre_3_signal4854.525879
Spike_AFFX-r2-Bs-lys_3-5-ratio1.275014
Spike_AFFX-r2-Bs-dap_M_signal330.205231
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio10.784683
Spike_AFFX-r2-Ec-bioB_avg-signal30.712229
Spike_AFFX-r2-Bs-thr_avg-signal125.129578
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1039.733887
Spike_AFFX-r2-Bs-phe_5_signal49.038181
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1163.417969
RawQ2.475724
Spike_AFFX-r2-Bs-lys_5_signal52.307938
Signal(A)4.488273
%A34.379658
Signal(All)148.355179
Corner+Avg:190,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal217.052780
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal106.144440
Spike_AFFX-r2-Ec-bioD_avg-signal1101.575928
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P63.445858
Spike_AFFX-r2-Bs-lys_avg-signal55.269363
Spike_AFFX-r2-P1-cre_avg-signal4371.390137
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.174485
Spike_AFFX-r2-Bs-thr_3_signal262.387665
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal71.472878
Spike_AFFX-r2-Bs-dap_3_signal699.691956
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.118957
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal258.143097
#M496
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal66.693352
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.840823
Spike_AFFX-r2-Bs-thr_M_signal88.671425
Signal(P)230.843155
Spike_AFFX-r2-Bs-phe_3-5-ratio2.164526
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:9103,Count:9
Spike_AFFX-r2-P1-cre_5_signal3888.254639
Spike_AFFX-r2-Bs-phe_M_detectionP
#A7842
Signal(M)16.174479
BackgroundAvg:59.80,Std:0.34,Min:58.7,Max:60.7
Spike_AFFX-r2-Ec-bioC_avg-signal237.597931
Spike_AFFX-r2-Bs-dap_avg-signal370.161865
Spike_AFFX-r2-Bs-dap_3-5-ratio8.682291
Spike_AFFX-r2-Ec-bioB_5_signal29.949139
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.530926
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp2_pgy1_IP_rep1_ATH1

Add to Slide Selection

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Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp2_pgy1_IP_rep1_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp2_pgy1_IP_rep1_ATH1
Organism: Arabidopsis thaliana
Alias: pgy1-1 pgy3-1 pgy3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY1 At2g27530,PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy1-1 pgy3-1 pgy3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy1-1 and pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then crossed and taken to F2 to produce homozygous PGY1 pgy3-1 PGY3p::PGY3-FLAG (wild-type control for PGY1) and pgy1-1 pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.571947
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.143441
Spike_AFFX-r2-Ec-bioB_3-5-ratio0.871275
Spike_AFFX-r2-Bs-dap_5_signal84.253525
NoiseAvg:3.02,Std:0.07,Min:2.8,Max:3.2
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal27.819651
#P13917
Spike_AFFX-r2-Bs-phe_M_signal75.232101
Corner-Avg:12105,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal36.772453
Spike_AFFX-r2-Ec-bioB_3_signal36.447250
Spike_AFFX-r2-Bs-lys_M_signal38.610931
Spike_AFFX-r2-P1-cre_3_signal4669.253906
Spike_AFFX-r2-Bs-lys_3-5-ratio1.500911
Spike_AFFX-r2-Bs-dap_M_signal419.915436
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio11.180563
Spike_AFFX-r2-Ec-bioB_avg-signal38.350590
Spike_AFFX-r2-Bs-thr_avg-signal145.663803
Spike_AFFX-r2-Bs-thr_5_detectionP
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal1266.762695
Spike_AFFX-r2-Bs-phe_5_signal69.379799
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal1519.007813
RawQ2.681310
Spike_AFFX-r2-Bs-lys_5_signal68.515465
Signal(A)5.363886
%A36.641823
Signal(All)151.581085
Corner+Avg:203,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal312.158295
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal99.411697
Spike_AFFX-r2-Ec-bioD_avg-signal1392.885254
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P61.012714
Spike_AFFX-r2-Bs-lys_avg-signal69.987335
Spike_AFFX-r2-P1-cre_avg-signal4376.381836
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.345463
Spike_AFFX-r2-Bs-thr_3_signal311.039368
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal81.341194
Spike_AFFX-r2-Bs-dap_3_signal747.777405
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.199126
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal355.483002
#M535
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal102.835609
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio0.878124
Spike_AFFX-r2-Bs-thr_M_signal98.132378
Signal(P)244.507065
Spike_AFFX-r2-Bs-phe_3-5-ratio1.432862
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:10291,Count:9
Spike_AFFX-r2-P1-cre_5_signal4083.510254
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8358
Signal(M)18.558056
BackgroundAvg:64.88,Std:0.79,Min:63.1,Max:66.6
Spike_AFFX-r2-Ec-bioC_avg-signal333.820648
Spike_AFFX-r2-Bs-dap_avg-signal417.315460
Spike_AFFX-r2-Bs-dap_3-5-ratio8.875325
Spike_AFFX-r2-Ec-bioB_5_signal41.832073
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.571947
NF1.000000
HZ4
Tau0.015000

Slide: Pignocchi_exp1_wt_TOT_rep3_ATH1

Add to Slide Selection

Get the data!

Summary BioSource 1 Information Extraction / Labelling Protocol for Source 1 Information Slide Information Hybridisation Analysis Information for "Affymetrix MAS 5.0 Scaling Protocol" normalised data.

Summary Information

This slide has had 1 extract(s) hybridised to it (1-colour).

  • Extract 1 ("Pignocchi_exp1_wt_TOT_rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

The slide used was "Affymetrix ATH1 Arabidopsis Genome Array".

Click on the options above for more information about this slide and its preparation.

Hybridisation Protocol Information

Name of Protocol: Affymetrix Antibody Amplification Protocol
Solution:
BufferEDTA, NaCl, Tween20 according to Affymetrix protocol
Water
Blocking Agent:
Herring Sperm DNA(Source: Promega)100pg/ml
Acetylated BSA(Source: Sigma-Aldrich)0.1mg/ml
Wash Procedure:
Step 1(Source: Affymetrix)10 cycles of 2 mixes/cycle with Non-Stringent buffer
Step 2(Source: Affymetrix)4 cycles of 15 mixes/cycle with Stringent buffer
Step 3(Source: Affymetrix)Stain the probe array for 10 minutes in Streptavaidin Phycoerythrin (SAPE) solution at 25 C
Step 4(Source: Affymetrix)10 cycles of 4 mixes/cycle with Non-Stringent buffer
Step 5(Source: Affymetrix)Stain the probe array for 10 minutes in antibody solution
Step 6(Source: Affymetrix)Stain the probe array for 10 minutes in SAPE solution at 25 C
Step 7(Source: Affymetrix)15 cycles of 4mixes/cycle with Non-Stringent buffer at 30 C
Step 8(Source: Affymetrix)Hold at 25 C
Quantity Used:
Amount15 microgram Fragmented cRNA
Time: 16 hours
Concentration: 50 pg/ml
Volume: 200 microlitres
Temperature: 45 C
Other Information:
Spike(Source: Affymetrix)Control Oligo B2 to 50pM for landing lights
Spike(Source: Affymetrix)20X Eukaryotic Hybridisation Controls (bioB (1.5pM, bioC 5pM, bioD 25pM, cre 100pM)
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

BioSource 1 Information

BioSource Name: Pignocchi_exp1_wt_TOT_rep3_ATH1
Organism: Arabidopsis thaliana
Alias: pgy3-1 PGY3-FLAG
Stock Code:
Genetic Background: Ler-0
Age: 7 days
Growth Conditions:
Stratification2 days 4 C in liquid medium, after sterilisation
SterilisationSterilised 100 µL of dry seeds using dichloroisocyanuric acid method: 1. Under the laminar flow hood add 1ml of the sterilisation to each tube of seeds and mix well by inverting the tube for 15 min (do not exceed 15 min). 2. Wash the seeds 3 X 1ml sterile water 3. Transfer the seeds to 50ml liquid medium using a “cut-off” P1000 Gilson tip. Sterilisation solution 50mg/ml Dichloroisocyanuric acid (aq) 1 ml Sterile water 3 ml 100% ethanol 4 ml
ProtocolSeedlings germinated and grown for 7 days in 50 ml ½ GM liquid medium (see below) under constant light, at 22 C, on an orbital shaker at 70-80 rpm. Yield after 7 days: 5-6 g fresh weight.
Substrate Sterilising Procedureautoclaving (120 C/15 min)
Plant Spacing100 ul dry seeds in 50 mL liquid medium
Temperature22 °C average, 22 °C day, 22 °C night
Humidity100 % average, 100 % day, 100 % night
MediumMurashige & Skoog basal salt mixture. Modifications: 1.25 mM MES pH 5.7 0.5% glucose 2.2g Murashige and Skoog salts + vitamins per 1L
Lighting(Source: Fluorescent manufactured by Phillips TC-D 70 W 830. Intensity: 80 µEinsteins. sqm-1. sec-1µEinsteins. Wavelength: White)Constant light
Developmental Stage:
Growth Stage(Source: Paradigm Genetics)1.05
Genetic Variation: EMS induced mutation mutation in PGY3 At3g25520,PGY3:PGY3-FLAG (transgene)
Tissue: whole plant
Additional Organism Information:
Sample DescriptionGeneration of pgy3-1 PGY3-FLAG: A 2.7 Kb genomic fragment encompassing At3g25520 (PGY3) was cloned into the binary vector pGWB10 to generate PGY3p::PGY3-FLAG. This construct was transformed into pgy3-1 (Pinon et al., 2008) using Agrobacterium mediated transformation (Clough and Bent, 1998). Transformants were selected based on complementation of pgy3-1 and on expression of PGY3-FLAG detected by western blot, then taken to F2 to produce pgy3-1 PGY3p::PGY3-FLAG. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. Pinon, V., Etchells, J.P., Rossignol, P., Collier, S.A., Arroyo, J.M., Martienssen, R.A., and Byrne, M.E. (2008). Three PIGGYBACK genes that specifically influence leaf patterning encode ribosomal proteins. Development 135, 1315-1324.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen / Trizol
Method:
ProtocolQiagen RNAeasy kit protocol
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Labelling Protocol Information

Name of Protocol: Biotin Labelled cRNA using Affymetrix Enzo Kit
Amount Labelled: Unknown
Label Name: Biotin
Labelling Method: In-vitro transcription method
Other Information:
ProtocolSourceAffymetrix GeneChip Technical Analysis Manual

Slide Information

Date Hybridised:

Array Design Information

Slide Design Name: Affymetrix ATH1 Arabidopsis Genome Array
Array Type: Affymetrix Gene Chip
Number of Spots on the Array: 22810

Scanning Protocol Information

Name: Affymetrix MAS 5.0 Standard Scanning
Width of Image Produced (Pixels): 4733
Height of Image Produced (Pixels): 4733
X Resolution (microns): 3
Y Resolution (microns): 3
Scanner Name: Agilent 2500A GeneArray Scanner
Software Name: Affymetrix Micro Array Suite 5.0

Image Analysis Information

Scaling Factor:0.980506
Name: Affymetrix MAS 5.0 Standard Image Analysis
Software Name: Affymetrix Microarray Analysis Suite 5.0

Normalisation Information

Strategy Name: Affymetrix MAS 5.0 Scaling Protocol
Algorithm: The top 2% and bottom 2% of signal intensities are excluded, then the mean is calculated. The original signal values are scaled such that the mean is made equal to 100.
Control Elements:
none
Normalisation Protocol Parameters:
Spike_AFFX-r2-P1-cre_3-5-ratio1.197343
Spike_AFFX-r2-Ec-bioB_3-5-ratio1.206144
Spike_AFFX-r2-Bs-dap_5_signal15.558301
NoiseAvg:2.35,Std:0.07,Min:2.2,Max:2.6
Control DirectionAntisense
Spike_AFFX-r2-Ec-bioB_5_detectionP
Spike_AFFX-r2-Ec-bioB_3_detectionP
Spike_AFFX-r2-Bs-thr_5_signal11.841911
#P13578
Spike_AFFX-r2-Bs-phe_M_signal32.737713
Corner-Avg:14388,Count:32
Spike_AFFX-r2-P1-cre_3_detectionP
Spike_AFFX-r2-Ec-bioB_M_signal226.389542
Spike_AFFX-r2-Ec-bioB_3_signal198.903793
Spike_AFFX-r2-Bs-lys_M_signal34.699547
Spike_AFFX-r2-P1-cre_3_signal9460.745117
Spike_AFFX-r2-Bs-lys_3-5-ratio1.627198
Spike_AFFX-r2-Bs-dap_M_signal150.237213
Spike_AFFX-r2-Bs-thr_M_detectionP
Probe Pair Threshold1
Spike_AFFX-r2-Bs-thr_3-5-ratio14.585730
Spike_AFFX-r2-Ec-bioB_avg-signal196.734055
Spike_AFFX-r2-Bs-thr_avg-signal81.487755
Spike_AFFX-r2-Bs-thr_5_detectionA
Spike_AFFX-r2-Bs-dap_3_detectionP
Spike_AFFX-r2-Ec-bioD_5_signal2155.400391
Spike_AFFX-r2-Bs-phe_5_signal26.369020
Spike_AFFX-r2-P1-cre_5_detectionP
Spike_AFFX-r2-Ec-bioD_3_signal2539.206299
RawQ2.456891
Spike_AFFX-r2-Bs-lys_5_signal27.374586
Signal(A)6.342141
%A38.224464
Signal(All)158.504684
Corner+Avg:222,Count:32
Spike_AFFX-r2-Ec-bioD_5_detectionP
Spike_AFFX-r2-Ec-bioC_3_signal743.782837
Spike_AFFX-r2-Bs-lys_3_detectionP
Spike_AFFX-r2-Bs-phe_3_signal167.275284
Spike_AFFX-r2-Ec-bioD_avg-signal2347.303223
Spike_AFFX-r2-Bs-dap_5_detectionP
Spike_AFFX-r2-Bs-phe_5_detectionP
%P59.526524
Spike_AFFX-r2-Bs-lys_avg-signal35.539333
Spike_AFFX-r2-P1-cre_avg-signal8681.095703
Spike_AFFX-r2-Ec-bioB_M_detectionP
Spike_AFFX-r2-Bs-lys_M_detectionP
#Probe Sets Exceeding Probe Pair Threshold22810
%M2.249014
Spike_AFFX-r2-Bs-thr_3_signal172.722916
Spike_AFFX-r2-Ec-bioC_5_detectionP
Spike_AFFX-r2-Bs-phe_3_detectionP
Spike_AFFX-r2-Ec-bioC_3_detectionP
Spike_AFFX-r2-Bs-phe_avg-signal75.460670
Spike_AFFX-r2-Bs-dap_3_signal637.046326
Spike_AFFX-r2-Ec-bioD_3-5-ratio1.178067
Spike_AFFX-r2-Ec-bioD_3_detectionP
Spike_AFFX-r2-Ec-bioC_5_signal674.058838
#M513
Spike_AFFX-r2-Bs-lys_5_detectionP
Spike_AFFX-r2-Bs-lys_3_signal44.543869
Spike_AFFX-r2-Bs-dap_M_detectionP
Spike_AFFX-r2-Ec-bioC_3-5-ratio1.103439
Spike_AFFX-r2-Bs-thr_M_signal59.898415
Signal(P)261.416046
Spike_AFFX-r2-Bs-phe_3-5-ratio6.343629
Spike_AFFX-r2-Bs-thr_3_detectionP
Central-Avg:11392,Count:9
Spike_AFFX-r2-P1-cre_5_signal7901.446289
Spike_AFFX-r2-Bs-phe_M_detectionP
#A8719
Signal(M)20.834131
BackgroundAvg:57.40,Std:0.48,Min:56.6,Max:58.6
Spike_AFFX-r2-Ec-bioC_avg-signal708.920837
Spike_AFFX-r2-Bs-dap_avg-signal267.613953
Spike_AFFX-r2-Bs-dap_3-5-ratio40.945751
Spike_AFFX-r2-Ec-bioB_5_signal164.908844
ScaleMaskAll
BG2
VZ4
TGT100
Alpha10.040000
Alpha20.060000
SF0.980506
NF1.000000
HZ4
Tau0.015000


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