|
| Status |
Public on Oct 01, 2023 |
| Title |
MuSC vehilce treated, biological rep. 3 |
| Sample type |
RNA |
| |
|
| Source name |
MuSC from mouse #2 treated with vehicle
|
| Organism |
Mus musculus |
| Characteristics |
treatment: control
tissue: adult muscle stem cells
mouse strain: C57BL/6J
|
| Treatment protocol |
Cells were stimulated with a pulse of 1 h duration with 100 ng/ml BMP6 or only with vehicle used for reconstitution of BMP in case of controls.
|
| Growth protocol |
Muscle stem cells were isolated from muscle of female 6-8-week-old mice, cultured on Matrigel® for expansion over 2 days. In order to limit the effct of BMPs in the culture medium (e.g. from serum supplements), cells were cultured prior to the BMP stimulation for 6 h in serum-free medium containing BMP soluble receptor (recombinant mouse BMPR-IA/ALK3-Fc Chimera, R&D Systems, 437-MR) in a concentration of 200 ng/ml. Cells were then stimulated for 1 h with a pulse of of 100 ng/ml BMP4 or BMP6 or only with vehicle used for reconstitution of BMP in the case of controls.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted. Cells were lysed directly in their dish, homogenized, and stored at -80 deg C for subsequent RNA isolation. Lysis (RLT buffer+10% βME), homogenization (QIAshredder, Qiagen), and total RNA extraction were done according to the manual of the RNeasy Micro kit (Qiagen).
|
| Label |
biotin
|
| Label protocol |
Affymetrix GeneChip™ WT PLUS Reagent Kit was used to generate sense-strand target (ST) cDNA. Briefly, first-cycle cDNA was generated from total RNA using a reverse transcription priming method that specifically primes non-ribosomal RNA from the sample, including both poly(A) and nonpoly(A) mRNA. This was then used to generate antisense cRNA by in vitro transcription. The second-cycle ST cDNA was synthesized from cRNA using random primers resulting in a product that contained dUTP at a fixed ratio relative to dTTP. Uracil-DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE1) were then used to recognize and fragment the cDNA at the unnatural dUTP residues. Fragmented cDNA was then biotinylated by terminal deoxynucleotidyl transferase (TdT). Fragmentation and biotinylation were done with the Affymetrix GeneChip® WT Terminal Labeling Kit.
|
| |
|
| Hybridization protocol |
Biotinylated cDNA was hybridized to the array at 45°C, 60 rpm for 16 h. The hybridized array was stained with streptavidin phycoerythrin conjugate using the Affymetrix GeneChip® Fluidics Station 450. Hybridization and staining steps were done with the Affymetrix GeneChip® Hybridization, Wash, and Stain Kit.
|
| Scan protocol |
The arrays were scanned with the Affymetrix GeneChip® Scanner 3000 G7.
|
| Description |
Gene expression data from control treated MuSC
|
| Data processing |
The R package oligo (PMID: 20688976) was used for quality control and pre-processing. Raw CEL files were background corrected, normalized and summarized using the RMA algorithm (PMID: 12582260) with mogene10st_Mm_ENSG_21.0.0 (December 2016) as a custom Chip Description File (CDF) from the BrainArray project (PMID: 16284200). The Ensembl IDs were mapped to gene symbols using Ensembl BioMart and the mouse reference assembly GRCm38.p4. Software versions: R v3.3.0, Bioconductor v 3.3
|
| |
|
| Submission date |
Mar 04, 2019 |
| Last update date |
Oct 01, 2023 |
| Contact name |
Markus Schuelke |
| E-mail(s) |
[email protected]
|
| Phone |
++49 30 4505 66112
|
| Organization name |
Charite
|
| Department |
Neuropediatrics
|
| Lab |
Schuelke lab
|
| Street address |
Augustenburger Platz 1
|
| City |
Berlin |
| State/province |
Berlin |
| ZIP/Postal code |
13353 |
| Country |
Germany |
| |
|
| Platform ID |
GPL26250 |
| Series (1) |
| GSE127798 |
The effect of BMP4/6 stimulation on adult muscle stem cells (MuSC) |
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