|
| Status |
Public on Jun 16, 2014 |
| Title |
Ctrl KD, PGC1a OV, biol. rep. 2 |
| Sample type |
RNA |
| |
|
| Source name |
C2C12 cells with Ctrl KD, PGC1a OV
|
| Organism |
Mus musculus |
| Characteristics |
differentiation stage: C2C12 myotubes
phenotype: PGC-1alpha over-expression
treatment: non-targeting siRNA
|
| Treatment protocol |
The siRNAs for the individual knockdown of the following genes were purchased from Dharmacon (Fisher Scientific) and used to transfect myotubes: Fos, Jun, Atf3 and the non-targeting siRNA pool (control). The transfection was performed using the siRNAs at a final concentration of 50nM and DharmaFECT1 transfection reagent according to the Thermo Scientific DharmaFECT Transfection Reagents siRNA Transfection Protocol. 24h after the transfection with siRNAs for Fos, Jun and Atf3 knockdown, the cells were cultivated in the presence of the PGC-1alpha expressing adenovirus for 48h. The control transfection cells (previously transfected with the non-targeting siRNA pool) were cultivated in the presence of either GFP or PGC-1alpha adenovirus.
|
| Growth protocol |
C2C12 cells were grown to 90% confluence in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 Units/ml penicillin and 100ug/ml streptomycin. The differentiation to myotubes was induced by changing the medium to DMEM supplemented with 2% horse serum for 72h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA was extracted using Trizol® and according to the Trizol® Reagent RNA extraction protocol.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cDNA were prepared according to the WT Expression Kit (Ambion) followed by the WT Terminal Labeling and Hybridization Labeling Kit (Affymetrix) from 270ng total RNA
|
| |
|
| Hybridization protocol |
3 µg of fragmented cRNA were hybridized for 17 hr at 45°C on GeneChip Mouse Gene 1.0 ST Array. GeneChips were washed and stained in the Fluidics Station 450 (Affymetrix) under FS450_0002 protocol, using the Hybridization Wash and Stain Kit (Affymetrix)
|
| Scan protocol |
The GeneChips were scanned with an Affymetrix GeneChip Scanner 3000 7G
|
| Data processing |
Raw probe intensities were corrected for background and unspecific binding using the Bioconductor package “affy”. Probes were classified as expressed or non-expressed by using the “Mclust” R package and, after removal of non-expressed probes, the intensity values were quantile normalized across all samples. Using mapping of the probes to the UCSC collection of mouse mRNAs, probes were then associated to a comprehensive collection of mouse promoters available from the SwissRegulon database (Pachkov et al., 2013). The log2 expression level of a given promoter was calculated as the weighted average of the expression levels of all probes associated to it.
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| |
|
| Submission date |
Sep 26, 2013 |
| Last update date |
Jun 17, 2014 |
| Contact name |
Christoph Handschin |
| Organization name |
Biozentrum, University of Basel
|
| Street address |
Spitalstrasse 41
|
| City |
Basel |
| ZIP/Postal code |
4056 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL10740 |
| Series (2) |
| GSE51190 |
Transcriptional network analysis in muscle reveals AP-1 as a partner of PGC-1α in the regulation of the hypoxic gene program [microarray: kD_AP1] |
| GSE51191 |
Transcriptional network analysis in muscle reveals AP-1 as a partner of PGC-1α in the regulation of the hypoxic gene program |
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