|
| Status |
Public on Apr 23, 2013 |
| Title |
myb2 KO vs Col0 time 24hpi biorep B |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
myb2 t1
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: myb2 KO
time: 24hpi
|
| Treatment protocol |
B. cinerea pepper strain spores (Denby et al., 2004) were prepared and Arabidopsis leaves treated as described in Windram et al., Plant Cell 2012 Sep;24(9):3530-57. PMID: 23023172. Col-0, myb2 and myb108 leaves were inoculated with 4-6 (depending on leaf size) evenly spaced 10μl droplets of B. cinerea spores. Infected leaves were harvested to provide 4 biological replicates. Samples for the comparison between myb108 and Col-0 were harvested at 26 and 30 hpi. Samples for the comparison between myb2 knockout line and Col-0 or myb2 knockout line and Col-0 were harvested at 26 and 30 hpi or 24 and 30 hpi respectively.
|
| Growth protocol |
Arabidopsis plants Col-0, myb2 and myb108 were grown under a 16:8 hr light:dark cycle (lights on 04:00 to 20:00) at 20°C, 60% humidity and light intensity of 100 μmol photons.m-2.s-1. Arabidopsis seed was stratified for three days in 0.1% agar at 4°C before sowing onto Arabidopsis soil mix (Scotts Levingtons F2s compost:sand:fine grade vermiculite in a ratio of 6:1:1). The myb2, myb108 lines were T-DNA insertion lines Salk_045455, Salk_024059 respectively (obtained from the Nottingham Arabidopsis Seed Centre).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from four individual leaves from each sampled time point (arbitrarily labelled as biological replicates A, B, C and D) using TRIzol reagent (Invitrogen), purified with RNeasy columns (Qiagen) and amplified using the MessageAmp II aRNA Amplification kit (Ambion) in accordance with the kit protocol with a single round of amplification.
|
| Label |
Cy3
|
| Label protocol |
Cy3 and Cy5 labelled cDNA probes were prepared by reverse transcribing 5µg of aRNA with Cy3- or Cy5- dCTP (GE Healthcare) and a modified dNTP mix (10mM each dATP, dGTP and dTTP; 2mM dCTP) using random primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with the inclusion of RNase inhibitor (RNaseOUT, Invitrogen) and DTT. Labelled probes were purified using QiaQuick PCR Purification columns (Qiagen), freeze-dried, and resuspended in 50µl hybridization buffer (25% formamide, 5xSSC, 0.1% SDS, 0.5µg/µl yeast tRNA [Invitrogen]). Four biological replicates were pooled and labelled twice with each dye giving four technical replicates. Comparisons were made pairwise between WT and mutant under each condition.
|
| |
|
| Channel 2 |
| Source name |
Col0 t1
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: Col0 WT
time: 24hpi
|
| Treatment protocol |
B. cinerea pepper strain spores (Denby et al., 2004) were prepared and Arabidopsis leaves treated as described in Windram et al., Plant Cell 2012 Sep;24(9):3530-57. PMID: 23023172. Col-0, myb2 and myb108 leaves were inoculated with 4-6 (depending on leaf size) evenly spaced 10μl droplets of B. cinerea spores. Infected leaves were harvested to provide 4 biological replicates. Samples for the comparison between myb108 and Col-0 were harvested at 26 and 30 hpi. Samples for the comparison between myb2 knockout line and Col-0 or myb2 knockout line and Col-0 were harvested at 26 and 30 hpi or 24 and 30 hpi respectively.
|
| Growth protocol |
Arabidopsis plants Col-0, myb2 and myb108 were grown under a 16:8 hr light:dark cycle (lights on 04:00 to 20:00) at 20°C, 60% humidity and light intensity of 100 μmol photons.m-2.s-1. Arabidopsis seed was stratified for three days in 0.1% agar at 4°C before sowing onto Arabidopsis soil mix (Scotts Levingtons F2s compost:sand:fine grade vermiculite in a ratio of 6:1:1). The myb2, myb108 lines were T-DNA insertion lines Salk_045455, Salk_024059 respectively (obtained from the Nottingham Arabidopsis Seed Centre).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from four individual leaves from each sampled time point (arbitrarily labelled as biological replicates A, B, C and D) using TRIzol reagent (Invitrogen), purified with RNeasy columns (Qiagen) and amplified using the MessageAmp II aRNA Amplification kit (Ambion) in accordance with the kit protocol with a single round of amplification.
|
| Label |
Cy5
|
| Label protocol |
Cy3 and Cy5 labelled cDNA probes were prepared by reverse transcribing 5µg of aRNA with Cy3- or Cy5- dCTP (GE Healthcare) and a modified dNTP mix (10mM each dATP, dGTP and dTTP; 2mM dCTP) using random primers (Invitrogen) and SuperScript II reverse transcriptase (Invitrogen), with the inclusion of RNase inhibitor (RNaseOUT, Invitrogen) and DTT. Labelled probes were purified using QiaQuick PCR Purification columns (Qiagen), freeze-dried, and resuspended in 50µl hybridization buffer (25% formamide, 5xSSC, 0.1% SDS, 0.5µg/µl yeast tRNA [Invitrogen]). Four biological replicates were pooled and labelled twice with each dye giving four technical replicates. Comparisons were made pairwise between WT and mutant under each condition.
|
| |
|
| |
| Hybridization protocol |
The microarray experiments were carried out using the CATMA (version 4) microarray (Allemeersh et al., 2005; http://www.catma.org). Labelled samples were hybridized to slides overnight at 42oC.
|
| Scan protocol |
Following hybridization, slides were washed and scanned using an Affymetrix 428 array scanner at 532nm (Cy3) and 635nm (Cy5).
|
| Description |
myb2 KO vs Col0 WT at 24hpi
|
| Data processing |
Scanned data were quantified using Imagene 7.5.0 software (BioDiscovery, Inc.).
Analysis of expression differences between Col-0 and myb2 and Col-0 and myb108 under each condition was performed using the R Bioconductor package limmaGUI (Wettenhall and Smyth, 2004). Raw data were normalized within arrays using a PrintTip lowess transformation and normalized between arrays using the quantile-normalization. The data were fitted to a linear model using a least squares method. P-values were adjusted for multiple testing using Benjamini and Hochberg method to control the false discovery rate (Benjamini and Hochberg, 1995).
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| |
|
| Submission date |
Mar 28, 2013 |
| Last update date |
Apr 23, 2013 |
| Contact name |
Jonathan David Moore |
| E-mail(s) |
jonathan.moore@tgac.ac.uk
|
| Organization name |
The Genome Analysis Centre
|
| Department |
Platforms and Pipelines
|
| Street address |
Norwich Research Park
|
| City |
Norwich |
| ZIP/Postal code |
NR4 7UH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16132 |
| Series (2) |
| GSE45594 |
A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves (Botrytis cinerea infection). |
| GSE46318 |
A Local Regulatory Network Around Three NAC Transcription Factors in Stress Responses and Senescence in Arabidopsis leaves |
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