|
| Status |
Public on Aug 22, 2011 |
| Title |
C2C12_ShB |
| Sample type |
SRA |
| |
|
| Source name |
C2C12 myoblasts
|
| Organism |
Mus musculus |
| Characteristics |
cell type: C2C12 cell derived myotubes
|
| Treatment protocol |
C2C12 cells were infected with lentiviral vectors and selected in the continuous presence of puromycin. Infected cell populations were then differentiated for 7 days in DMEM medium with 2% horse serum.
|
| Growth protocol |
C2C12 cells were cultured in DMEM with 10% FCS and differentiated by replacing growth medium with differentiation medium containing 2% horse serum
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The mRNA-seq libraries were prepared following the Illumina protocol with some modifications. Briefly, mRNA was purified from total RNA using oligo-dT magnetic beads and fragmented using divalent cations at 95C for 5 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers. This was followed by second strand cDNA synthesis using Polymerase I and RNase H. The double strand cDNA fragments were blunted, phosphorylated and ligated to single-end adapter dimers follwed by PCR amplification (30 sec at 98C; [10 sec at 98C, 30 sec at 65C, 30 sec at 72C] x 13 cycles; 5 min at 72C). After PCR amplification, surplus PCR primers and dimer adapters were removed by purification using AMPure beads (Agencourt Biosciences Corporation). Size selection was performed by electrophoresis on a 2% agarose gel and DNA fragments in the range of ~250-350bp were excised and purified using QIAquick Gel Extraction Kit (Qiagen). DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent). The libraries were loaded in the flowcell at 6pM concentration and clusters generated and sequenced on the Illumina Genome Analyzer IIx as single-end 72 base reads
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
C2C12 cells expressing a shRNA to silence TEAD4 expression
|
| Data processing |
Base calling : Image analysis and base calling were performed using the Illumina Pipeline version 1.6.
Alignment : Reads were mapped to reference genome mm9/NCBI37 using Tophat v1.0.12.
Quantification : Quantification of gene expression was done using Cufflinks v0.8.2 and annotations from Ensembl release 57. For each transcript the resulting FPKM were converted into raw read counts and these counts were added for each gene locus.
Normalization : Data normalization was performed with the DESeq Bioconductor package.
|
| |
|
| Submission date |
Mar 08, 2011 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Celine Keime |
| Organization name |
IGBMC (CNRS/INSERM/UDS)
|
| Street address |
1 rue Laurent Fries
|
| City |
Illkirch |
| ZIP/Postal code |
67404 |
| Country |
France |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE27845 |
TEAD4 regulated genes in differentiated C2C12 myoblasts |
|
| Relations |
| BioSample |
SAMN02196988 |
