|
| Status |
Public on Nov 19, 2013 |
| Title |
8W T. salsuginea rosette leaves non acclimated replicate 1 |
| Sample type |
RNA |
| |
|
| Source name |
8-week-old rosette leaves, non-acclimated
|
| Organism |
Eutrema salsugineum |
| Characteristics |
tissue: rosette leaves
treatment: non-acclimated
age: 8-week
species: Thellungiella salsuginea
cultivar: Yukon
|
| Treatment protocol |
Soil grown 6-week-old Col-0 plants were cold acclimated at 4°C for 2 weeks. Soil grown 8-week-old Yukon plants were cold acclimated at 4°C for 2 weeks.
|
| Growth protocol |
Col-0 seeds were stratified at 4°C for 1 week and grown on soil at 20°C for 3 weeks under 8 hrs light/16 hrs dark cycle at growth chamber. Col-0 plants were continued grown at 20°C for 2 weeks under 16 hrs light/8 hrs dark cycle at greenhouse. Yukon seeds were stratified at 4°C for 1 week and grown on soil at 20°C for 8 weeks under 16 hrs light/8 hrs dark cycle at greenhouse.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated and on-column DNA digestion was performed with the RNeasy Plant mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the Low Input Quick Amp Labeling (Agilent) 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 55 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 55 ml of 2x 2x GEx Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to a custom Gene Expression Agilent Microarray (G2514F; DesignID 031554) 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 (G2565CA) using one color scan setting for AgilentHD_GX for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5µm, Dye channel is set to Green and Green PMT is set to 100%, Tiff 20bit).
|
| Description |
Gene expression for non acclimated Yukon rosette leaves. RNA was hybridized on custom made Agilent Thellungiella salsuginea expression array
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.5.1.1 (Agilent) using default parameters (protocol GE1_105_Dec08 and Grid: 031554_D_F_20101207) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Nov 08, 2012 |
| Last update date |
Nov 19, 2013 |
| Contact name |
Ellen Zuther |
| E-mail(s) |
Zuther@mpimp-golm.mpg.de
|
| Phone |
+49 331 567 8253
|
| Organization name |
MPI of Molecular Plant Physiology
|
| Department |
Transcript Profiling
|
| Street address |
Am Muehlenberg 1
|
| City |
Potsdam |
| ZIP/Postal code |
D-14476 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16263 |
| Series (1) |
| GSE42156 |
Transcriptome sequencing and microarray design for functional genomics in the extremophile Arabidopsis relative Thellungiella salsuginea |
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