GSM6617506: M. musculus, TA, PBS, scRNA-seq; Mus musculus; RNA-Seq
Tibialis anterior muscle from 8-week-old mice were collected 5 days-post CTX or sham (PBS) injury (n = 5 biological replicates for each group). After manual dissociation of muscle tissue from blood vessels, fat, and connective tissue, samples were mulched in dissociation media (10mg/ml Collagenase D [Roche], 4.8U/ml Dispase II [Roche], and 10% horse serum in high-glucose DMEM) with fine scissors and incubated at 37°C for 45 minutes with agitation. Collagenase/dispase was inactivated with FBS, and the cells were filtered through a 40µm strainer and centrifuged for 10 minutes at 1000g at 4°C, washed in PBS, and underwent red blood cell lysis (Qiagen) and myofiber debris removal (Miltenyi Biotec, 130-109-398) using commercial kits following the manufacturer's instructions. Cell viability was >90% based on trypan blue staining in all samples. Biological replicates were then pooled and fixed in ice-cold methanol for 15 minutes on ice and stored at -80°C until single-cell RNA-sequencing. Single-cell cDNA libraries were prepared using 10x Genomics methanol fixation protocol (CG000136 rev. E).