|
| Status |
Public on Jan 01, 2015 |
| Title |
myoblasts-control siRNA-biological repl 01 |
| Sample type |
RNA |
| |
|
| Source name |
mock-treated proliferating myoblasts
|
| Organism |
Mus musculus |
| Characteristics |
tissue: skeletal muscle
cell type: satellite cells-derived myoblasts
|
| Treatment protocol |
Proliferating myoblasts were transfected with siRNA by using Lipofectamine 2000 and Optimem (Life Technologies) following manufacturer's protocol
|
| Growth protocol |
satellite cells-derived myoblasts were cultured on collagen-coated dishes in Ham's-F10 nutrient mix supplemented with 20% FBS and 2.5ng/ul basic FGF
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed by using the TRIzol Reagent (Life Technologies) according to manufacturer's instructions. 50 ng of total RNA was reverse transcribed following the Ovation PicoSL WTA System (Nugen). The resulting double strand cDNA was used for amplification based on SPIA technology. After purification according to Nugen protocol, 5ug of single strand DNA was used for generation of Sens Target DNA using Ovation Exon Module kit (Nugen).
|
| Label |
biotin
|
| Label protocol |
2.5μg of Sens Target DNA were fragmented and biotin labelled using Encore Biotin Module kit (Nugen).
|
| |
|
| Hybridization protocol |
After control of fragmentation using Bioanalyzer 2100, cDNA was hybridized to GeneChip® Mouse Gene 1.0 ST (Affymetrix) at 45°C for 17 hours. After overnight hybridization, chips are washed on the fluidic station FS450 following specific protocols (Affymetrix)
|
| Scan protocol |
GeneChips were scanned using the affymetrix GCS3000 7G system
|
| Data processing |
The scanned images are then analyzed with Expression Console software (Affymetrix) to obtain raw data and metrics for Quality Controls. The observations of some of these metrics and the study of the distribution of raw data show no outlier experiment. Affymetrix probe-set data were normalized using the robust multi-array average (RMA) method using R
|
| |
|
| Submission date |
May 22, 2014 |
| Last update date |
Jan 01, 2015 |
| Contact name |
Fabien Le Grand |
| E-mail(s) |
fabien.le-grand@inserm.fr
|
| Phone |
0033144412436
|
| Organization name |
Institut Cochin
|
| Department |
DRC
|
| Street address |
24 rue du Fg Saint Jacques
|
| City |
Paris |
| ZIP/Postal code |
75014 |
| Country |
France |
| |
|
| Platform ID |
GPL17777 |
| Series (1) |
| GSE57898 |
Transcriptomic analysis of APC knockdown in proliferating primary myoblasts |
|
