|
| Status |
Public on Nov 10, 2014 |
| Title |
MPC_28_D4_1_4 |
| Sample type |
RNA |
| |
|
| Source name |
Primary myotubes differentiated for 4 day replicate 3
|
| Organism |
Mus musculus |
| Characteristics |
gender: Male
strain: C57BL/6J
cell type: Primary myotubes
growth protocol: differentiation conditions at day 4
|
| Treatment protocol |
To induce myotube formation, confluent proliferating primary myoblasts were grown on matrigel coated culture dishes (Basement Membrane Matrix, Becton and Dickinson) and switched to differentiation medium next day (DM: DMEM plus 2% horse serum, 100 U/ml penicillin and100 µg/ml streptomycin (Life technologies)) for 1,2 and 4 days.
|
| Growth protocol |
Primary myoblast were cultured in growth medium (GM: Ham’s F-10 plus 20% FBS, 5 ng/ml bFGF, 100 U/ml penicillin and 100 µg/ml streptomycin) on 100 mg Collagen I rat tail (Becton and Dickinson) coated tissue culture dishes
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100ng of total RNA using the LowInputQuick Amp Labeling kit - Agilent 5190-2305 according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
After fragmentation, 600 ng of labeled cRNA from each sample was hybridized in in situ hybridization oven (Agilent) for 17 h at 65ºC and washed during 1 min at rt in Gene Expression Wash Buffer 1 (Agilent) and 1 min at 37 ºC with Gene Expression Wahs buffer 2 (Agilent).
|
| Scan protocol |
Scanned on an Agilent G2539A scanner at 3um resolution and 100%PMT. The intensity data of each individual hybridization were extracted and the quality was assessed with the Feature Extraction software 10.7 (Agilent).
|
| Description |
Muscle precursor cells in differentiation conditions at day 4
|
| Data processing |
Raw data was corrected for background noise using the normexp method. Quantile normalization was applied to assure comparability across samples.
|
| |
|
| Submission date |
Nov 10, 2014 |
| Last update date |
Nov 10, 2014 |
| Contact name |
Miguel A Peinado |
| E-mail(s) |
mpeinado@igtp.cat
|
| Organization name |
IGTP
|
| Street address |
Ctra. de Can Ruti, camí de les escoles, s/n
|
| City |
Badalona |
| State/province |
Barcelona |
| ZIP/Postal code |
08916 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13912 |
| Series (1) |
| GSE63136 |
Deconstruction of DNA methylation patterns during myogenesis reveals Myf5 super-enhancer hypomethylation in the establishment of the muscle lineage |
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