title: GSM6678961: human, Semitendinosus, 01, scRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: Semitendinosus muscle from consenting young patients (n = 5; 2 males, 3 females, mean age = 23.4 years) undergoing routine orthopedic surgery for anterior cruciate ligament repair was immediately placed into preservation media (20% FBS in DMEM) following biopsy and transported to the laboratory for immediate single-cell analysis. Upon arrival, muscle sections were washed with PBS, any associated tendons, ligaments, adipose tissue, and blood vessels removed, and tissue weighed. Muscle was then mulched for ~5 mins in a few drops of HBSS using fine surgical scissors. Dissociation media consisting of collagenase D (10 mg/mL, Roche), and 2.4 U/mL dispase II (Sigma) was then added to the samples and the samples were incubated at 37°C for 45 minutes, with periodic agitation. Ten volumes of PBS were added, and the cell-containing solution was filtered through a 70 µm strainer, and centrifuged at 1100 x g for 10 mins. RBCs were lysed and myofiber debris removed using commercially available kits (Qiagen, 158904, Miltenyi Biotec, 130109398, respectively) as per the manufacturer's instructions. Trypan blue viability tests (VWR, K940) were performed on all samples and a minimum of 80% of the cells in each sample were found to be viable. Cells were then fixed in ice-cold methanol for 15 minutes, and stored at -80°C. Cells were recoved from methanol by centrifugation at 1000 x g for 5 mins at 4°C , and then rehydrated using a wash-resuspension buffer, consisting of 0.04% BSA, 1mM DTT, and 0.2 U/µL RNAse inhibitor in a 3x saline-sodium citrate buffer (in nuclease-free water). All samples were then combined and loaded into the 10x Chromium controller and scRNA-seq performed.