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Experiment: Photoreceptor control of shoot meristem activity and leaf initiation

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-426

When Arabidopsis seedlings are grown in the dark, their shoot meristem remains in a repressed state and does not develop leaves. The tight control of leaf development by light provides the opportunity to analyse a fundamental plant developmental process in its very early stages. We have observed that activation of photoreceptors in dark-grown seedlings leads to a rapid and dramatic increase in mitotic activity in and around the shoot meristem. Cotyledons also exhibit an induction of cell cycle activity by light, but this consists of endoreduplication and occurs later. Many other processes take place upon induction by light, including the development of the photosynthetic apparatus and the synthesis of sunscreens, but these processes are expected to take place in both the leaf primordia and the cotyledons. We have used this information to carry out a whole transcriptome analysis of shoot meristem activation and early stages of leaf development by light. For this we dissected the shoot apical regions from seedlings grown in the dark or 1, 2, 6, 24, 48 or 72 h after transfer to light. We also dissected cotyledons from equivalent seedlings grown in the dark or 1 or 6 h after transfer to light. From these samples we obtained RNA used for hybridisations against the ATH1 GenChip.

About the Experimenter

Name:Dr Enrique Lopez-Juez
Head of Lab Name:Dr Enrique Lopez-Juez
Lab:
Address:School of Biological Sciences
Royal Holloway, University of London
Egham
Surrey
Postcode: TW20 0EX
Country: UK
 
Telephone Number: 01784443951
Fax Number: 01784 414224

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: time_series_design; growth_condition_design; organism_part_comparison_design
Number of Slides:16
 
Experimental Parameters:
parameter timepoint
parameter organism_part
parameter change_light
Quality Control Measures Taken:
References:
 
Other Information:
GEO AccessionGSE19261

Slides in this Experiment

Hybridisation Set: Lopez-Juez: Photoreceptor control of shoot meristem activity and leaf initiation_genome

Slide: Lopez-Juez_1-1_Shoot-apex-dark_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 ("Lopez-Juez_1-1_Shoot-apex-dark_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: Lopez-Juez_1-1_Shoot-apex-dark_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 0hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time the seedlings were harvested under a green safelight.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region
Additional Organism Information:
sample description3 day old, dark-grown seedling. Approximately 10 mm long, folded cotyledons.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:0.814458
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:
RawQ3.41
NoiseAvg:4.79,Stdev:1.63,Max:12.8,Min:3.3
BackgroundAvg:86.03,Stdev:8.53,Max:100.7,Min:60.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.814458
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-2_Shoot-apex-dark_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 ("Lopez-Juez_1-2_Shoot-apex-dark_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: Lopez-Juez_1-2_Shoot-apex-dark_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 0hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time the seedlings were harvested under a green safelight.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region
Additional Organism Information:
sample description3 day old, dark-grown seedling. Approximately 10 mm long, folded cotyledons.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:2.4177
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:
RawQ2.33
NoiseAvg:3.12,Stdev:0.12,Max:3.4,Min:2.8
BackgroundAvg:63.61,Stdev:0.64,Max:66.1,Min:61.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF2.417700
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-3_Shoot-apex-1hr-light_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 ("Lopez-Juez_1-3_Shoot-apex-1hr-light_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: Lopez-Juez_1-3_Shoot-apex-1hr-light_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 1hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transfered back to white light for 1 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description3 day old, dark-grown seedling, after 1 h in the light. Approximately 10 mm long, folded cotyledons.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:0.651248
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:
RawQ3.94
NoiseAvg:4.25,Stdev:0.15,Max:4.8,Min:3.8
BackgroundAvg:104.93,Stdev:1.76,Max:110.9,Min:99.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.651248
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-4_Shoot-apex-1hr-light_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 ("Lopez-Juez_1-4_Shoot-apex-1hr-light_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: Lopez-Juez_1-4_Shoot-apex-1hr-light_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 1hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transfered back to white light for 1 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description3 day old, dark-grown seedling, after 1 h in the light. Approximately 10 mm long, folded cotyledons.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:0.65591
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:
RawQ3.54
NoiseAvg:3.60,Stdev:0.14,Max:4.0,Min:3.1
BackgroundAvg:96.93,Stdev:0.86,Max:100.0,Min:94.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.655910
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-5_Shoot-apex-2hr-light_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 ("Lopez-Juez_1-5_Shoot-apex-2hr-light_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: Lopez-Juez_1-5_Shoot-apex-2hr-light_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 2hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transfered back to white light for 2 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description3 day old, dark-grown seedling, after 2 h in the light. Approximately 10 mm long, folded cotyledons.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:0.756691
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:
RawQ3.18
NoiseAvg:3.53,Stdev:0.29,Max:5.2,Min:3.1
BackgroundAvg:76.63,Stdev:1.92,Max:82.4,Min:71.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.756691
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-6_Shoot-apex-6hr-light_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 ("Lopez-Juez_1-6_Shoot-apex-6hr-light_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: Lopez-Juez_1-6_Shoot-apex-6hr-light_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 6hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transfered back to white light for 6 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description3 day old, dark-grown seedling, after 6 h in the light. Approximately 10 mm long, cotyledons unfolding and starting to green.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:0.957308
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:
RawQ3.26
NoiseAvg:3.21,Stdev:0.15,Max:3.8,Min:2.8
BackgroundAvg:79.85,Stdev:1.94,Max:86.5,Min:75.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.957308
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-7_Shoot-apex-6hr-light_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 ("Lopez-Juez_1-7_Shoot-apex-6hr-light_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: Lopez-Juez_1-7_Shoot-apex-6hr-light_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 6hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transfered back to white light for 6 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description3 day old, dark-grown seedling, after 6 h in the light. Approximately 10 mm long, cotyledons unfolding and starting to green.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:1.283761
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:
RawQ2.68
NoiseAvg:3.13,Stdev:0.78,Max:6.5,Min:2.3
BackgroundAvg:64.64,Stdev:4.07,Max:70.6,Min:49.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.283761
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-8_Shoot-apex-24hr-light_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 ("Lopez-Juez_1-8_Shoot-apex-24hr-light_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: Lopez-Juez_1-8_Shoot-apex-24hr-light_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 24hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transfered back to white light for 24 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description4 day old seedling (3 days in the dark, one in the light). Approximately 10 mm long, cotyledons unfolded and green.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:0.915619
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:
RawQ3.18
NoiseAvg:3.37,Stdev:0.22,Max:4.0,Min:3.0
BackgroundAvg:78.53,Stdev:1.67,Max:83.1,Min:74.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.915619
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-9_Shoot-apex-48hr-light_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 ("Lopez-Juez_1-9_Shoot-apex-48hr-light_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: Lopez-Juez_1-9_Shoot-apex-48hr-light_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 48hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transfered back to white light for 48 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region and developing leaf primordia
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description5 day old seedling (3 days in the dark, two in the light). Approximately 10 mm long, cotyledons unfolded and green, leaf primordia visible.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:1.219697
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:
RawQ3.22
NoiseAvg:3.13,Stdev:0.17,Max:3.6,Min:2.8
BackgroundAvg:78.79,Stdev:2.07,Max:84.7,Min:73.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.219697
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-10_Shoot-apex-72hr-light_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 ("Lopez-Juez_1-10_Shoot-apex-72hr-light_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: Lopez-Juez_1-10_Shoot-apex-72hr-light_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 72hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transfered back to white light for 72 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: shoot apical meristem region and developing leaf primordia
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description6 day old seedling (3 days in the dark, 3 in the light). Approximately 10 mm long, cotyledons unfolded and green, very young leaves emerging.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:1.319209
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:
RawQ3.39
NoiseAvg:3.29,Stdev:0.16,Max:3.7,Min:2.8
BackgroundAvg:82.39,Stdev:1.92,Max:87.7,Min:77.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.319209
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-11_Cotyledons-dark_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 ("Lopez-Juez_1-11_Cotyledons-dark_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: Lopez-Juez_1-11_Cotyledons-dark_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 0hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were harvested.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: Cotyledon, with both the cotyledon petiole and the cotyledon tip removed
Additional Organism Information:
sample description3 day old seedling. Approximately 10 mm long, cotyledons folded.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:1.084113
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:
RawQ3.43
NoiseAvg:3.63,Stdev:0.27,Max:4.4,Min:3.2
BackgroundAvg:94.92,Stdev:5.87,Max:110.7,Min:82.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.084113
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-12_Cotyledons-dark_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 ("Lopez-Juez_1-12_Cotyledons-dark_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: Lopez-Juez_1-12_Cotyledons-dark_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 0hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were harvested.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: Cotyledon, with both the cotyledon petiole and the cotyledon tip removed
Additional Organism Information:
sample description3 day old dark-grown seedling. Approximately 10 mm long, cotyledons folded.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:1.087991
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:
RawQ2.69
NoiseAvg:2.58,Stdev:0.14,Max:3.1,Min:2.2
BackgroundAvg:66.43,Stdev:1.14,Max:69.3,Min:63.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.087991
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-13_Cotyledons-1hr-light_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 ("Lopez-Juez_1-13_Cotyledons-1hr-light_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: Lopez-Juez_1-13_Cotyledons-1hr-light_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 1hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transferred back to white light for 1 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: Cotyledon, with both the cotyledon petiole and the cotyledon tip removed
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description3 day old dark-grown seedling, after only 1 h in the light. Approximately 10 mm long, cotyledons folded.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:1.035366
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:
RawQ3.16
NoiseAvg:2.95,Stdev:0.14,Max:3.4,Min:2.5
BackgroundAvg:83.73,Stdev:2.34,Max:88.7,Min:78.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.035366
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-14_Cotyledons-1hr-light_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 ("Lopez-Juez_1-14_Cotyledons-1hr-light_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: Lopez-Juez_1-14_Cotyledons-1hr-light_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 1hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transferred back to white light for 1 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: Cotyledon, with both the cotyledon petiole and the cotyledon tip removed
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description3 day old dark-grown seedling, after only 1 h in the light. Approximately 10 mm long, cotyledons folded.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:0.554748
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:
RawQ3.40
NoiseAvg:3.74,Stdev:0.22,Max:4.4,Min:3.3
BackgroundAvg:91.75,Stdev:4.65,Max:102.8,Min:80.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.554748
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-15_Cotyledons-6hr-light_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 ("Lopez-Juez_1-15_Cotyledons-6hr-light_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: Lopez-Juez_1-15_Cotyledons-6hr-light_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 6hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transferred back to white light for 6 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: Cotyledon, with both the cotyledon petiole and the cotyledon tip removed
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description3 day old dark-grown seedling, after 6 h in the light. Approximately 10 mm long, cotyledons unfolding and starting to green.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:1.299473
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:
RawQ2.87
NoiseAvg:2.74,Stdev:0.11,Max:3.1,Min:2.5
BackgroundAvg:72.85,Stdev:1.48,Max:76.8,Min:69.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF1.299473
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Lopez-Juez_1-16_Cotyledons-6hr-light_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 ("Lopez-Juez_1-16_Cotyledons-6hr-light_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: Lopez-Juez_1-16_Cotyledons-6hr-light_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Landsberg erecta
Stock Code: N1642
Age: 6hr timepoint
Growth Conditions:
stratificationImbibed (after sterilisation) in 0.1% agar for 3 days at 4 C in the dark.
sterilisationWith ethanol, then dilute bleach, then rinses with sterile water
Growth protocolSeedlings were raised by exposing stratified seeds, sown on agar-solidified Murashige and Skoog medium (without sucrose), for 30 min to 100 micromol m-2 s-1 fluorescent cool white light, on horizontal plates as described (Vinti et al.; The Plant Journal 24(6), 883-894, 2000), to stimulate germination. The plates were placed in a dark incubator at 21 C for 3 days, at which time they were transferred back to white light for 6 h before harvesting.
LocationGrowth chamber
Growth substrateAgar
Average humidity50-80
Growth mediumMurashige and Skoog basal salt mixture
Growth medium modificationsMES, pH 5.7
Developmental Stage:
developmental stage(Source: Boyes paradigm)0.7
Tissue: Cotyledon, with both the cotyledon petiole and the cotyledon tip removed
in vivo Treatment:
TreamentLight (transfer from the dark)
Additional Organism Information:
sample description3 day old dark-grown seedling, after 6 h in the light. Approximately 10 mm long, cotyledons unfolding and starting to green.
Other Information:

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: RiboPure Kit (Ambion)
Method:
ProtocolSeedlings were immersed in RNAlater (Ambion) for 1 h (the harvesting and immersion being under green safelight), and stored at 4°C. Within one week seedlings were dissected on a chilled platform under a stereomicroscope, by slicing below the meristem and at the base of each cotyledon (?meristem? or M samples) or near the base and tip of each cotyledon (?cotyledon? or C samples), before flash-freezing and storing at -80°C. Only one tissue was obtained per seedling and the number of seedlings per sample ranged from ca.1500 (72 h samples) to over 4000 (0 h samples). Each sample contained material from multiple plates and several separate experiments. RNA was extracted using an RiboPure kit (Ambion), essentially according to manufacturer's instructions.

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:0.834978
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:
RawQ2.96
NoiseAvg:2.82,Stdev:0.12,Max:3.3,Min:2.5
BackgroundAvg:74.08,Stdev:1.29,Max:77.7,Min:70.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.834978
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


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