|
| Status |
Public on Feb 25, 2013 |
| Title |
cold_JZ_rcf1_3 |
| Sample type |
RNA |
| |
|
| Source name |
rcf1-1, cold stress 4°C 12 h
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype/variation: rcf1-1 mutant
tissue: whole seedlings
Stage: Fourtheen-day-old
|
| Treatment protocol |
Fourteen-day-old Arabidopsis wild type and rcf1-1 seedlings grown on MS agar medium were subjected to cold stress at 4°C for 12 h.
|
| Growth protocol |
Arabidopsis seedlings on Murashige and Skoog (MS) medium agar plates (1x MS salts, 2% sucrose, 0.6% agar, pH 5.7) were routinely grown under cool, white light (~120 μmol m-2 s-1) at 21 ± 1℃ with a 16-h-light/8-h-dark photoperiod.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Trizol (Invitrogen) and phenol:chloroform:isoamyl (25:24:1, PH 5.2) and precipitated with NaAc and Ethanol. RNA was then washed with 70% Ethanol and dissolved in water.
|
| Label |
Biotin
|
| Label protocol |
dsDNA was fragmented and labeled using the GeneChip® WT Double-Stranded cDNA Synthesis Kit.
|
| |
|
| Hybridization protocol |
Probes were hybridized to Arabidopsis Tiling 1.0R arrays for 14 h at 42°C. Arrays were washed using a Fluidics Station 450, wash protocol FS450_0001.
|
| Scan protocol |
Tiling arrays were scanned using a GeneChip® Scanner 3000 7G.
|
| Description |
A firefly luciferase reporter gene driven by the cold stress-responsive CBF2 promoter (-1500 bp to -1 bp upstream of the transcription start site) was introduced into Arabidopsis plants in the Columbia glabrous1 (gl1) background.
rcf1-1 under cold stress
|
| Data processing |
We first re-mapped the tiling array probes to the Arabidopsis genome (TAIR10) using SOAP2 and kept only probes that perfectly matched to a unique position in the genome for subsequent analyses. We then created a custom Chip Definition File (CDF) using the probe mapping result and used the aroma.affymetrix framework to quantile-normalize raw tiling array data. To identify retained introns, we first calculated the log2 signal intensity for each annotated intron (TAIR10) based on the trimmed-mean of signal intensities from all mapped probes. Introns with less than three mapped probes or low expression (log2 expression value < 5 in all samples) were not further considered. We then used the SAM algorithm to identify introns with significantly elevated expression in the rcf1-1 mutant samples relative to the wild type. A FDR (False Discovery Rate) of 0.05 was used as the significance cutoff.
|
| |
|
| Submission date |
Oct 05, 2012 |
| Last update date |
Feb 25, 2013 |
| Contact name |
Jianhua Zhu |
| Phone |
3014050920
|
| Organization name |
University of Maryland
|
| Department |
Plant Science and Landscape Architecture
|
| Street address |
2102 Plant Sciences Building
|
| City |
College Park |
| State/province |
MD |
| ZIP/Postal code |
20742 |
| Country |
USA |
| |
|
| Platform ID |
GPL10977 |
| Series (1) |
| GSE41378 |
Genome-wide identification of mis-spliced/intron-retained transcripts in Arabidopsis thaliana rcf1-1 mutant plants under cold stress as determined by tiling array analysis. |
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