|
| Status |
Public on Aug 11, 2014 |
| Title |
3T3_control_untreated_replicate3 |
| Sample type |
RNA |
| |
|
| Source name |
Swiss 3T3 fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
cell line: 3T3
cell type: fibroblasts
|
| Treatment protocol |
Serum-straved (72 h) mouse 3T3 fibroblasts we treated with 188.5 nM of anisomycin (Sigma) for 1, 3 and 6h.
|
| Growth protocol |
Swiss 3T3 fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (vol/vol) fetal calf serum (FCS) in the presence of Penicillin and Streptomycin. The cells were serum-deprived for 72 h using DMEM containing 0.2% FCS (vol/vol).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated using Trizol (Invitrogen) according to manufacturer'instructures.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using the Low Input Quick Amp Labelling kit, One-color, according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). cRNA was quantified with the NanoDrop ND-1000 UV-VIS 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, 55µl of 2x GEx Agilent Hybridization Buffer HI-RPM was added to the fragmentation mixture and hybridized to Agilent Whole Mouse 4x44k expression array (G2514F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with Agilent GE Wash Buffer 1 and 1 minute with 37°C Agilent GE Wash Buffer 2, followed by drying for 1 min with acetonitril.
|
| Scan protocol |
Slides were scanned with Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 4x44k array slides (Scan Area 61x21.6 mm, Scan resolution 5µm, Dye channel is set to Green).
|
| Description |
Gene expression after 72h starvation in the medium containing 0.2% FCS (replicate3)
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software version 10.7.1.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 014868_D_F_20100617) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Mar 04, 2014 |
| Last update date |
Aug 12, 2014 |
| Contact name |
Anna Sawicka |
| E-mail(s) |
anna.sawicka@mpibpc.mpg.de
|
| Organization name |
Max Planck Institure for Biophysical Chemistry
|
| Department |
Dep. of Molecular Biology
|
| Lab |
Cramer
|
| Street address |
Am Fassberg 11
|
| City |
Goettingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
| |
|
| Platform ID |
GPL4134 |
| Series (2) |
| GSE55554 |
Transcriptional response to stress in serum deprived mouse fibroblasts |
| GSE55784 |
Genome-wide analysis of stress response in mouse fibroblasts |
|
