|
| Status |
Public on Dec 23, 2013 |
| Title |
cotyledon_dark_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
cotyledon, dark, replicate 2
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
organ: cotyledon
light treatment: dark
|
| Treatment protocol |
Seeds on plate were stratified at 4℃ for 3 days and exposed to white light (100 umol m-2 s-1) for 1h at 22℃ to initiate germination before being transferred to continuous darkness at 22℃ for 4 day. After sampling of etiolated seedlings in the dark, seedlings on plates were exposed to continuous white light (100 umol m-2 s-1) for 1 and 6h. Prior to sampling, de-etiolated seedlings were divided into cotyledons, hypocotyls and roots, and organ samples were harvested and frozen immediately in liquid nitrogen and stored at -80℃.
|
| Growth protocol |
Arabidopsis thaliana ecotype Col-0 plants were used in this study. For sampling etiolated and de-etiolated seedlings, seeds were surface-sterilized in a 70% ethanol briefly and placed directly into a 30% Clorox with 0.1% Triton X-100 for 10 min and rinsed five times with sterilized water. Seeds were resuspended in a sterilized 0.15% (w/v) agarose and then sown in rows onto Murashige and Skoog (MS; MB biomedical, LLC, 263324) medium with 1% sucrose, Myo-inositol (Sigma, I3011), MES (Sigma, M8250) and 0.8% Bactoagar.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted and treated with DNase I using RNeasy Plant mini kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
We performed RNA labeling, hybridization and scaning using protocols according to the Agilent's instructions. Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent).
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to ATH NAT array 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), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner using one color scan setting for 8x60k array slides.
|
| Data processing |
Raw data of processed signal intensities are obtained by analysis using Feature Extraction Software (Agilent) with default parameters. Standard QC analysis, background normalization and quantile normalization were performed using GeneSpring GX Software (Agilent) with default parameters.
|
| |
|
| Submission date |
Jul 30, 2013 |
| Last update date |
Dec 25, 2013 |
| Contact name |
Huan Wang |
| E-mail(s) |
hwang01@rockefeller.edu
|
| Organization name |
The Rockefeller University
|
| Lab |
Chua Nam-Hai
|
| Street address |
1230 York Avenue
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL17515 |
| Series (1) |
| GSE49382 |
Dynamic changes of Arabidopsis cis-NAT pairs during de-etiolation |
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