|
| Status |
Public on Feb 21, 2013 |
| Title |
ced2 p6h 2 |
| Sample type |
RNA |
| |
|
| Source name |
CED2 PEG treatment for 6hr
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: gl1-Col0
genotype/variation: ced2 mutant
tissue: whole seedlings
age: 14d
treated with: 40% PEG solution for 6 h
|
| Treatment protocol |
14 d old seedlings of wild-type and ced2 were transferred to 40% PEG solution for 6 h.
|
| Growth protocol |
wild-type and ced2 seedlings were grown on MS plates for 14 d at 22°C with 16 h light and 8 h darkness.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted with Trizol reagent (Invitrogen) and pretreated with RNase-free DNase I (NEB), then cleaned using the RNeasy Plant Mini Kit (Qiagen). For each of two biological replicates, the whole seedlings from twenty plants were pooled to make a single RNA sample.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.3 ug RNA using the One-Color Low Input Quick Amp Labeling Kit, one-color (Agilent, 5190-2305) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.65 ug of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 55µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Arabidopsis (V4) Gene Expression Microarray, 4x44K (G2519F-021169) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent) and air dried before scanning.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um (Double pass), Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after 6 h treatment in 40%PEG solution
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters (protocol GE1-107_SEP09 and Grid: 021169_D_F-20110203) to obtain background subtracted and multiplicative (Spatial) detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
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| |
|
| Submission date |
Feb 20, 2013 |
| Last update date |
Feb 21, 2013 |
| Contact name |
zhenyu wang |
| E-mail(s) |
[email protected]
|
| Phone |
966028082661
|
| Organization name |
KAUST
|
| Street address |
PSGC
|
| City |
Thuwal |
| ZIP/Postal code |
23955-6900 |
| Country |
Saudi Arabia |
| |
|
| Platform ID |
GPL12621 |
| Series (1) |
| GSE44419 |
Expression data of WT (NCED3pro:LUC transgenic plants, gl1 Columbia background) and ced2 under osmotic stress |
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