|
| Status |
Public on Sep 04, 2017 |
| Title |
siP300_HPM_T24 diff |
| Sample type |
RNA |
| |
|
| Source name |
HPM transfected with sip300 at time 24h
|
| Organism |
Homo sapiens |
| Characteristics |
time: 24h of differentiation
cell type: Primary myoblasts
patient id: M38
|
| Treatment protocol |
Human Primary Myoblasts (HPM) were grown to confluence, transfectedor not with siRNA (either control, or CBP or CBP or CBP+p300) and induced 24h later for differentiation by decreasing serum concentration.
|
| Growth protocol |
myoblasts were plated in Petri dishes and cultured in growth medium containing Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 20% fetal bovine serum (GE Healthcare, PAA) 1% Ultroser G serum substitute (PALL life sciences) and 50μg/ml Gentamicin (Thermo Scientific) at 37°C in humidified atmosphere with 5% CO2. All experiments were carried out between P4 and P8 to avoid cell senescence. Myogenic differentiation of confluent cells was induced by changing growth medium to DMEM containing 2% FBS and 50μg/ml Gentamicin (differentiation medium).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted from cells using Aurum Total RNA Mini Kit (Bio-rad) according to manufacturer’s instructions
|
| Label |
biotin
|
| Label protocol |
Biotinylated single strand cDNA targets were prepared, starting from 150 ng of total RNA, using the Ambion WT Expression Kit and the Affymetrix GeneChip® WT Terminal Labeling Kit according to Affymetrix recommendations
|
| |
|
| Hybridization protocol |
Following fragmentation and end-labeling, 3 μg of cDNAs were hybridized for 16 h at 45oC on GeneChip® Human Gene 2.0 ST arrays (Affymetrix)
|
| Scan protocol |
The chips were washed and stained in the GeneChip® Fluidics Station 450 (Affymetrix) and scanned with the GeneChip® Scanner 3000 7G (Affymetrix) at a resolution of 0.7 µm
|
| Data processing |
Raw data (.CEL Intensity files) were extracted from the scanned images using the Affymetrix GeneChip® Command Console (AGCC) version 3.2.
CEL files were further processed with Affymetrix Expression Console software version 1.3.1 to calculate probe set signal intensities using Robust Multi-array Average (RMA) algorithms with default setings
|
| |
|
| Submission date |
Jan 24, 2017 |
| Last update date |
Sep 04, 2017 |
| Contact name |
Laurence Vandel |
| E-mail(s) |
laurence.vandel@univ-tlse3.fr
|
| Organization name |
CNRS
|
| Department |
CBD
|
| Street address |
118 ROute de Narbonne
|
| City |
Toulouse |
| ZIP/Postal code |
31062 |
| Country |
France |
| |
|
| Platform ID |
GPL16686 |
| Series (1) |
| GSE94006 |
Analysis of the common and respective roles of CBP and p300 co-activators in human primary myoblast differentiation. |
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