|
| Status |
Public on Jun 04, 2014 |
| Title |
shcontrol ESC rep2_A |
| Sample type |
RNA |
| |
|
| Source name |
ESC E14wt
|
| Organism |
Mus musculus |
| Characteristics |
transduction: shcontrol (non-target) expression vector
background: 129P2/OlaHsd
|
| Treatment protocol |
shRNA approaches were conducted with pLKO.1 puro shRNA vectors (Sigma-Aldrich, France) of the TRC2 library. For Ns1 KD the TRCN0000241466 shRNA clone and for Msl1 KD the TRCN0000241378 shRNA clone was used. In addition, the shRNA Non-Target Control (Product No. SHC002) was applied. Production of lentiviral particles as well as infection of mESCs was conducted according to the manufacturer’s protocol.
|
| Growth protocol |
Wild type male mESCs (E14.wt) were cultivated on 0.1% gelatin and CD1 feeder cells (37°C, 5% CO2) in DMEM (4.5g/L), 15% foetal calf serum, leukemia inhibiting factor, penicillin/streptomycin, L-glutamine and non-essential amino acids. To work under feeder-free conditions cells were treated with 1mg/ml Collagenase (GIBCO) and 2mg/ml Dispase (GIBCO) and cultivated for one passage without feeder cells. Experiments were conducted at passage 26-29.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cDNA targets were prepared, starting from 150 ng of total RNA, using the Ambion WT Expression Kit (Cat 4411974) and the Affymetrix GeneChip WT Terminal Labeling Kit (Cat 900671) according to Affymetrix recommendations.
|
| |
|
| Hybridization protocol |
Following fragmentation and end-labeling, 3 ug of cDNAs were hybridized on GeneChip Mouse Gene 2.0 ST arrays (Affymetrix) for whole-transcript expression profiles.
|
| Scan protocol |
Washed and stained chips were scanned with the GeneChip Scanner 3000 7G (Affymetrix) at a resolution of 0.7um.
|
| Description |
Experiment A: gene expression data from shcontrol ESCs
|
| Data processing |
Obtained raw data (.CEL intensity files) were processed with Affymetrix Expression Console software version 1.1 to calculate probe set signal intensities using Robust Multi-array Average (RMA) algorithms with default settings.
|
| |
|
| Submission date |
Apr 09, 2014 |
| Last update date |
Jun 05, 2014 |
| Contact name |
Sarina Ravens |
| Organization name |
IGBMC
|
| Street address |
1 Rue Laurent Fries
|
| City |
Illkirch |
| ZIP/Postal code |
67404 |
| Country |
France |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE56646 |
MOF-associated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation [array] |
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