title: GSM4221071: MuSCP2; Mus musculus; RNA-Seq
library_construction_protocol: Hindlimb muscles were dissected from mice following euthanization and kept in wash solution (Ham's F10 media supplemented with 10% horse serum). The muscles were then finely minced with a surgical scalpel and digested in 2 mg/ml Collagenase II (Worthington, 340 U/mg) for 90 min with agitation. Dissociated fibers were washed and further digested with shaking in 100 U/ml Collagenase II and 2 U/ml Dispase (Invitrogen) for 30 min in a volume of 10 ml, passed 10 times through a 20-G needle to release the associated satellite cells and then washed two more times and filtered through a 45-mm cell strainer. Mononuclear cells from each sample were resuspended in 500 ml wash solution, stained with VCAM1-biotin, CD31-APC, CD45- APC and Sca1-Pacific-Blue antibodies (Biolegend) using a 1:100 dilution at 4°C with gentle agitation for 30 min, washed and resuspended in 500 ml and then stained with Streptavidin-PE-Cy7 (1:100) at 4°C with gentle agitation for 15 min. The resulting preparation contains satellite cells (VCAM1+/CD31-/CD45-/Sca-), which can be separated from remaining cells and debris using flow cytometry. RNA was extracted with the Qiagen kit. Libraries for non-injured animals were prepared with the SMARTer stranded total RNA sample prep kit - low input mammalian, with ribosomal RNA depletion; libraries for injured animals were made with the TruSeq RNA library prep kit, after poly-A selection.
identifiers.namespace: GEO
identifiers.id: GSM4221071