|
| Status |
Public on Jun 02, 2015 |
| Title |
P. syringae pv tomato DC3000 hrpA- mutant 8 hours biorep A |
| Sample type |
RNA |
| |
|
| Source name |
P. syringae pv tomato DC3000 hrpA- mutant 8 hours biorep A
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
strain: Col0
treatment: Infected P. syringae pv tomato DC3000 hrpA- mutant
hours_since_infection: 8
tissue: leaf
|
| Growth protocol |
Arabidopsis plants Col-0 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).
|
| 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 or 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]).
|
| |
|
| Hybridization protocol |
The order in which the 288 slides were hybridized and scanned was randomised to minimise the impact on differences between samples of any potential variation in the processing conditions. According to this randomised experimental design combinations of 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).
Scanned data were quantified using Imagene 7.5.0 software (BioDiscovery, Inc.).
|
| Description |
Randomised loop design providing dye-swaps between treatments, time points and bioreps
|
| Data processing |
Quantified microarray data were Lowess normalized within pintip groups and within arrays, then variation due to arrays and dyes was removed by a random effects model, and averaged, in log space, using R/MAANOVA (MicroArray ANalysis Of VAriance) (Wu et al. 2003). Oligo sequences of CATMA array probes were mapped to individual mRNA sequences of transcripts from the TAIR9 genome assembly using BLASTn with e-value cutoff of 0.01. Additionally, alignments were filtered to exclude alignments shorter than 30bp or with less than 80% sequence identity.
|
| |
|
| Submission date |
Mar 21, 2014 |
| Last update date |
Jun 03, 2015 |
| 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 |
GPL10840 |
| Series (1) |
| GSE56094 |
Transcriptional dynamics driving MAMP-triggered immunity and pathogen effector-mediated immunosuppression in Arabidopsis leaves following infection with Pseudomonas syringae pv. tomato DC3000. |
|
