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Experiment: Oligogalacturonide treatment of Arabidopsis seedlings

Experiment Description

NASCArrays Experiment Reference Number: NASCARRAYS-409

To assess the contribution to defenses against necrotrophic fungal pathogens that may be mediated by recognition of oligogalacturonides (OGs), cell wall fragments released by the activity of fungal polygalacturonases, we treated seedlings with OGs and assayed transcript levels 1 and 6 hours after addition of OGs to culture medium. For each sample, approximately 30 seedlings were grown in shallow liquid MS medium for 10 days. Plants were then treated with either 200 ug/ml OGs or, for control plants, an equal volume of water. Three replicate samples were assayed for each treatment.

About the Experimenter

Name:Dr. Julia Dewdney
Head of Lab Name:Dr. Frederick Ausubel
Lab:
Institute: Massachusetts General Hospital
Address:Dept. of Molecular Biology
Massachusetts General Hospital
Simches 7700
185 Cambridge St.
Boston, MA
Postcode: 02114
Country: USA
 
Telephone Number: 617-643-3325
Fax Number: 617 643 3050

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; compound_based_treatment_design
Number of Slides:18
 
Experimental Parameters:
parametercompound_based_treatment
parameter timepoint
Quality Control Measures Taken:
References:
 
Other Information:

Slides in this Experiment

Hybridisation Set: Dewdney: Oligogalacturonide treatment of Arabidopsis seedlings_genome

Slide: Dewdney_1-1_Flg22-1h_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 ("Dewdney_1-1_Flg22-1h_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: Dewdney_1-1_Flg22-1h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 1hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentFlg22, final concentration 1 uM, was added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.655205
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:
RawQ1.53
NoiseAvg:2.30,Stdev:0.11,Max:2.7,Min:2.1
BackgroundAvg:37.23,Stdev:0.42,Max:38.8,Min:36.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.655205
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-2_Flg22-1h_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 ("Dewdney_1-2_Flg22-1h_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: Dewdney_1-2_Flg22-1h_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 1hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentFlg22, final concentration 1 uM, was added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.660241
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:7.52,Stdev:0.33,Max:9.1,Min:7.0
BackgroundAvg:91.89,Stdev:1.06,Max:95.5,Min:89.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.660241
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-3_Flg22-1h_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Dewdney_1-3_Flg22-1h_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Dewdney_1-3_Flg22-1h_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 1hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentFlg22, final concentration 1 uM, was added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.989721
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:
RawQ1.52
NoiseAvg:2.06,Stdev:0.08,Max:2.4,Min:1.9
BackgroundAvg:38.42,Stdev:0.50,Max:40.4,Min:37.3
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.989721
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-4_Flg22-3h_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 ("Dewdney_1-4_Flg22-3h_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: Dewdney_1-4_Flg22-3h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 3hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentFlg22, final concentration 1 uM, was added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.681552
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:
RawQ1.57
NoiseAvg:2.49,Stdev:0.10,Max:2.8,Min:2.3
BackgroundAvg:38.58,Stdev:0.56,Max:41.0,Min:37.7
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.681552
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-5_Flg22-3h_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 ("Dewdney_1-5_Flg22-3h_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: Dewdney_1-5_Flg22-3h_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 3hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentFlg22, final concentration 1 uM, was added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.551956
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:
RawQ1.73
NoiseAvg:2.62,Stdev:0.10,Max:2.9,Min:2.4
BackgroundAvg:41.96,Stdev:0.52,Max:44.0,Min:40.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.551956
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-6_Flg22-3h_Rep3_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Dewdney_1-6_Flg22-3h_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Dewdney_1-6_Flg22-3h_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 3hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentFlg22, final concentration 1 uM, was added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.857971
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:
RawQ1.59
NoiseAvg:2.17,Stdev:0.09,Max:2.5,Min:1.9
BackgroundAvg:41.33,Stdev:0.47,Max:42.8,Min:40.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.857971
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-7_H20-1h_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 ("Dewdney_1-7_H20-1h_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: Dewdney_1-7_H20-1h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 1hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentwater was added to the medium on day 10
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.770039
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:
RawQ1.60
NoiseAvg:2.41,Stdev:0.12,Max:2.8,Min:2.2
BackgroundAvg:39.01,Stdev:0.56,Max:40.6,Min:37.6
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.770039
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-8_H20-1h_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 ("Dewdney_1-8_H20-1h_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: Dewdney_1-8_H20-1h_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 1hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentwater was added to the medium on day 10
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.878251
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:
RawQ1.41
NoiseAvg:1.97,Stdev:0.12,Max:2.5,Min:1.8
BackgroundAvg:37.19,Stdev:0.55,Max:38.9,Min:36.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.878251
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-9_H20-1h_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Dewdney_1-9_H20-1h_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Dewdney_1-9_H20-1h_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 1hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentwater was added to the medium on day 10
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.50229
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:
RawQ1.68
NoiseAvg:2.64,Stdev:0.12,Max:3.1,Min:2.4
BackgroundAvg:39.28,Stdev:0.39,Max:41.0,Min:38.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.502290
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-10_H20-3h_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 ("Dewdney_1-10_H20-3h_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: Dewdney_1-10_H20-3h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 3hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentwater was added to the medium on day 10
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.548421
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:
RawQ1.70
NoiseAvg:2.76,Stdev:0.15,Max:3.4,Min:2.5
BackgroundAvg:40.10,Stdev:0.52,Max:41.5,Min:39.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.548421
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-11_H20-3h_Rep2_ATH1

Add to Slide Selection

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

Summary Information

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

  • Extract 1 ("Dewdney_1-11_H20-3h_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: Dewdney_1-11_H20-3h_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 3hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentwater was added to the medium on day 10
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.810864
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:
RawQ1.22
NoiseAvg:1.55,Stdev:0.08,Max:1.9,Min:1.4
BackgroundAvg:34.91,Stdev:0.26,Max:35.9,Min:34.4
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.810864
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-12_H20-3h_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Dewdney_1-12_H20-3h_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Dewdney_1-12_H20-3h_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 3hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatmentwater was added to the medium on day 10
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.594569
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:
RawQ1.56
NoiseAvg:2.55,Stdev:0.16,Max:3.0,Min:2.2
BackgroundAvg:39.03,Stdev:0.79,Max:41.1,Min:37.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.594569
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-13_OGs-1h_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 ("Dewdney_1-13_OGs-1h_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: Dewdney_1-13_OGs-1h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 1hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml OGs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatment50 ug/ml oligogalacturonides, degree of polymerization 10-15, were added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.525178
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:
RawQ3.20
NoiseAvg:7.85,Stdev:0.33,Max:9.1,Min:7.2
BackgroundAvg:80.00,Stdev:0.66,Max:81.8,Min:78.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.525178
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-14_OGs-1h_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 ("Dewdney_1-14_OGs-1h_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: Dewdney_1-14_OGs-1h_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 1hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatment50 ug/ml oligogalacturonides, degree of polymerization 10-15, were added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.549271
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:
RawQ2.99
NoiseAvg:6.02,Stdev:0.23,Max:6.9,Min:5.5
BackgroundAvg:70.10,Stdev:0.49,Max:71.1,Min:68.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.549271
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-15_OGs-1h_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Dewdney_1-15_OGs-1h_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Dewdney_1-15_OGs-1h_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 1hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatment50 ug/ml oligogalacturonides, degree of polymerization 10-15, were added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.848259
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:
RawQ2.30
NoiseAvg:3.32,Stdev:0.11,Max:3.8,Min:3.1
BackgroundAvg:56.30,Stdev:0.52,Max:57.7,Min:54.9
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.848259
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-16_OGs-3h_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 ("Dewdney_1-16_OGs-3h_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: Dewdney_1-16_OGs-3h_Rep1_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 3hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatment50 ug/ml oligogalacturonides, degree of polymerization 10-15, were added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.569415
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:
RawQ1.61
NoiseAvg:2.48,Stdev:0.12,Max:2.8,Min:2.2
BackgroundAvg:39.84,Stdev:0.46,Max:41.6,Min:39.1
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.569415
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-17_OGs-3h_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 ("Dewdney_1-17_OGs-3h_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: Dewdney_1-17_OGs-3h_Rep2_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 3hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatment50 ug/ml oligogalacturonides, degree of polymerization 10-15, were added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.586344
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:
RawQ1.45
NoiseAvg:2.32,Stdev:0.13,Max:2.6,Min:2.0
BackgroundAvg:36.03,Stdev:0.41,Max:37.3,Min:35.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.586344
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015

Slide: Dewdney_1-18_OGs-3h_Rep3_ATH1

Add to Slide Selection

Get the data!

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

Summary Information

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

  • Extract 1 ("Dewdney_1-18_OGs-3h_Rep3_ATH1") was made from "Arabidopsis thaliana" and labelled using "Biotin Labelled cRNA using Affymetrix Enzo Kit".

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

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

Hybridisation Protocol Information

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

BioSource 1 Information

BioSource Name: Dewdney_1-18_OGs-3h_Rep3_ATH1
Organism: Arabidopsis thaliana
Alias: Col-0
Stock Code:
Age: 3hr timepoint
Growth Conditions:
LocationGrowth Room
stratification3 days 4oC
sterilisation5% hypochlorite 10 minutes
Growth protocolApproximately 15 seedlings were placed in 1 ml liquid MS medium with B5 vitamins and 0.5% sucrose, in one well of 12-well culture plate. Seedlings were grown at 23C in 100 uE per square meter per second light provided by white fluorescent bulbs under long day conditions (16h light/8h dark). At eight days, the medium was replaced with fresh medium. At 10 days, seedlings were treated with either 50 ug/ml Ogs, 1 uM flg22, or an equivalent volume of water. Seedlings were harvested and frozen in liquid N2 at 1 and 3 hours post treatment.
growth substrate typeHydroponics
hydroponic constituentsPlants were not grown hydroponically, but there is no other option to select for plants grown in liquid medium. Seedlings were grown in a shallow layer of medium.
sterilisationfilter sterilization
average humidity100
average temperature23
Growth mediumMurashige and Skoog basal salt mixture
Developmental Stage:
Growth Stage10 day-old seedlings
Tissue: whole plant
in vivo Treatment:
treatment50 ug/ml oligogalacturonides, degree of polymerization 10-15, were added to the medium on day 10.
Other Information: Columbia, USA Seed stock maintained in lab for 20 years.

Protocols for BioSource 1

Extraction Protocol Information

Name of Protocol: Qiagen RNAeasy kit
Method:
ProtocolSeedlings harvested and snap-frozen in liquid nitrogen and RNA extracted as described in the Qiagen RNAeasy kit.
Type: total RNA
Amplification: Amplified during labelling process - see labelling protocol
Other Information:
ProtocolSourceSubmitter

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:0.66632
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:
RawQ1.62
NoiseAvg:2.47,Stdev:0.14,Max:3.0,Min:2.3
BackgroundAvg:40.71,Stdev:0.77,Max:43.6,Min:39.2
Gamma1H0.0025
Gamma2L0.003
Gamma2H0.003
TGT100
Perturbation1.1
SF0.666320
Alpha10.04
Alpha20.06
SFGeneAll
NF1.000000
BF
Gamma1L0.0025
Tau0.015


Problems? Comments? Suggestions? Contact the Affymetrix Team