|
| Status |
Public on Apr 21, 2015 |
| Title |
Postnatal P28, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
mouse abdominal, pectoral and intercostal muscle including skeletal muscle satellite cells, P28
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6J
genotype/variation: Pax3GFP/+
tissue: abdominal, pectoral and intercostal muscle including skeletal muscle satellite cells
age: postnatal day 28
|
| Treatment protocol |
Pax3GFP/+ mice of the appropriate age were detected by PCR and splotch white tag in the belly, and GFP confirmed under fluorescent stereoscope. Muscle samples were isolated from the trunk and digested in DMEM high glucose but no phenol red, 0.1% Trypsin, 0.1% Collagenase D and 0.01mg/mL DNaseI, and purified by filtration using 100µm and 40µm cell strainers. GFP cells were stained using propidium iodide to exclude dead cells and purified via FACS Aria II based on gating of GFP signal.
|
| Growth protocol |
Pax3GFP/+ mice were bred with C57BL/6J and the day where plugs were detected was considered as E0.5 (E, embryonic days).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy® Micro Kit (QIAGEN) RNA extraction protocol was used according to the manufacturer's instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the Encore Biotin Module (NuGEN) protocol from 100ng total RNA (http://www.nugen.com/nugen/index.cfm/support/product-resources/tech/encore-biotin-module-performance/). Single-stranded cDNA is fragmented and characterized by chemical and enzymatic methods leading to molecules of about 50 to 100 bases. Next, a biotin-labeled nucleotide is attached to the 3' end of each fragment.
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| |
|
| Hybridization protocol |
High-density oligonucleotide arrays containing 45,000 sets of oligonucleotide probes (25 mers) that cover all 30,000 genes encoded by the murine genome (Affymetrix Mouse Genome 430 2.0 Arrays, Ref 900495) were used for gene expression detection. Hybridization during 16 hours at 45°C in a rotary oven (Affymetrix), washing and staining (GeneChip® Fluidics Station 450) and scanning (GeneChip Scanner 3000) were carried out according to NuGEN and Affymetrix protocols.
|
| Scan protocol |
GeneChips were scanned using the GeneChip Scanner 3000.
|
| Description |
Sample name: P-1.B.2012
|
| Data processing |
Expression Console software (Affymetrix) was used for image analysis and to determine probe signal levels. The quality and statistical analysis of the data were made using the GeneSpring GX11 analysis software (Agilent Technologies).
Data needed to be pre-treated before final analysis to normalize two different sets of arrays (2006 or 2012, as indicated in the sample names).
Expression profiles were normalized in batch using the RMA algorithm (affy R package) yielding a (probe sets, samples) matrix. As the 11 samples were obtained by merging two series, the Combat algorithm (Johnson WE, et al. Biostatistics, 2007) was used to normalize the corresponding batch effect. Expression profiles were aggregated by Gene Symbol (mean across probe sets) using Affymetrix csv annotation file (na32 version).
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| |
|
| Submission date |
Feb 13, 2015 |
| Last update date |
Apr 21, 2015 |
| Contact name |
Sonia Alonso-Martin |
| E-mail(s) |
[email protected]
|
| Phone |
+33684321566
|
| Organization name |
Myology Research Center U974-INSERM - UPMC-Paris VI - Institut de Myologie
|
| Department |
Development and stem cells
|
| Lab |
Team 8 - Relaix
|
| Street address |
105 bd de l'Hôpital
|
| City |
Paris |
| ZIP/Postal code |
75013 |
| Country |
France |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE65927 |
Early postnatal expression data from mouse skeletal muscle stem cells |
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