title: GSM8791675: Sample 2, KO replicate 1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Dorsal root ganglia from segmental levels C2 and below were dissected and dissociated to obtain neurons for single cell RNA sequencing analysis. Sensory ganglia were dissociated in 40 units papain (Worthington LK003178), 4 mg/ml collagenase (Worthington LS004176), 10 mg/ml BSA, 1 mg/ml hyalurdonidase, 0.6 mg/ml DNase in DMEM:F12 (Gibco #11330-032) + 1% pen/strep + 12.5 mM glucose for 25 min at 37 °C. Digestion was quenched using 20 mg/ml ovomucoid (trypsin inhibitor), 20 mg/ml BSA in DMEM:F12 + 1% pen/strep + 12.5 mM glucose. Ganglia were gently triturated with fire-polished glass pipettes (opening diameter of approx. 150–200 μm). Neurons were then passed through a 70-μm filter to remove cell doublets and debris. Neurons were pelleted and washed 4 times in 20 mg/ml ovomucoid (trypsin inhibitor), 20 mg/ml BSA in DMEM:F12 + 1% pen/strep + 12.5 mM glucose followed by 2× washes with DMEM:F12 + 1% pen/strep + 12.5 mM glucose all at 4 °C. After washing, cells were resuspended in 45 μl of DMEM:F12 + 1% pen/strep + 12.5 mM glucose.  ~8000-10000 dissociated cells from DRG were loaded per 10x run (10x genomics chromium single cell kit, v3) library preparation were performed according to manufacturer's instructions (10x genomics chromium single cell kit, v3)