|
| Status |
Public on Feb 17, 2017 |
| Title |
rep2 siLuc D0 SPJH30 |
| Sample type |
SRA |
| |
|
| Source name |
Primary myoblasts
|
| Organism |
Mus musculus |
| Characteristics |
tissue: myoblasts
strain: C57/B6
|
| Treatment protocol |
Total RNA was extracted using Sigma total RNA extraction kit
|
| Growth protocol |
Both C2C12 cells and primary myoblasts were transfected with control and Tead1/Tead4 siRNA and following which cells were subjected to 2% HS medium in order to differentiate.RNA was isolated from non differentiated cells 24hrs after transfection and at day 3 &day 6 of differentiation. Samples from duplicate biological replicates were subjected to RNA-seq analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After isolation of total cellular RNA fromthe above 24 samples, libraries of template molecules suitable for high throughput DNA sequencing were created using “TruSeq™ RNA Sample Preparation v2 Kit” (Illumina). Briefly, mRNA was purified from 2 µg of total RNA using poly-T oligo-attached magnetic beads and fragmented using divalent cations at 94°C for 8 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers then the second strand of the cDNA was synthesized using DNA Polymerase I and RNase H. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single ‘A’ nucleotide was added to the 3’ ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C). Then surplus PCR primers were removed by purification using AMPure XP beads (Agencourt Biosciences Corporation). DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Base calling : Image analysis and base calling were performed using RTA 1.17.21.3 and CASAVA 1.8.2
Alignment : Reads were mapped to mm9 assembly of mouse genome using Tophat v2.0.10 and bowtie2 v2.1.0 aligner
Quantification of gene expression was performed using HTSeq v0.6.1 (Anders et al., 2014) using gene annotations from Ensembl release 67.
Comparisons of interest were performed using the method proposed by Love et al (2014) and implemented in the DESeq2 Bioconductor library (DESeq2 v1.8.1), taking into account the batch, treatment and day effects
Genome_build: mm9
Supplementary_files_format_and_content: The processed data file is a tabulated text with the following columns : Ensembl gene id, Gene name and one column for each sample containing the normalized read counts (normalization was done using the size factors calculated with DESeq2 v1.8.1 Bioconductor package).
|
| |
|
| Submission date |
Jun 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Shilpy Joshi |
| E-mail(s) |
shilpy.joshi@gmail.com
|
| Organization name |
IGBMC
|
| Street address |
1 Rue laurent Fries
|
| City |
Illkirch |
| ZIP/Postal code |
67404 |
| Country |
France |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE82192 |
Specific and redundant roles of TEAD transcription factors in C2C12 cell and primary myoblast differentiation (RNA-Seq) |
| GSE82193 |
Specific and redundant roles of TEAD transcription factors in C2C12 cell and primary myoblast differentiation |
|
| Relations |
| BioSample |
SAMN05199441 |
| SRA |
SRX1817477 |
