GSM8353872: ACL non injured quadricep biopsy 2; Homo sapiens; RNA-Seq
Nuclei were isolated from a portion (~35 mg) of frozen quadriceps muscle obtained from participants before and 7d post-Sx. Muscle was mechanically minced with sterile scissors and dounced 25 times in homogenization buffer (10 mM HEPES, 60 mM KCl, 100 mM spermidine, 150 µM spermine tetrahydrochloride, 2 mM EDTA, 500 µM EGTA, 5 mM MgCl2, 5% sucrose in 50 mL of ultra-pure water) to liberate nuclei. Nuclear suspensions were passed through sequential 70 µm and 40 µm filters, spun at 500g x 5 min and resuspended in 300 µL of resuspension buffer (2% BSA in PBS with 1 µL SUPERase RNase Inhibitor (#AM2696, ThermoFisher). DAPI (1mg/ml, #D3571, ThermoFisher) was added to the cell suspension prior to sorting with a Fluorescent Activated Cell Sorter (SY3200, Sony Biotech, San Jose, CA) and gated for DAPI positive nuclei Isolated nuclei were then loaded onto a 10x chromium controller for barcode labelling. snRNA-seq libraries were generated using Chromium Next GEM Single Cell 3' HT Reagent kits v3.1 sn-RNA seq