GSM6753877: DRG, control,  timepoint 0, scRNAseq 10; Mus musculus; RNA-Seq
C57BL/6N mice (8-10 wk, Charles River, Sweden) were sacrificed with overdosage of isoflurane. Spines were removed and fresh cervical and lumbar dorsal root ganglia (DRGs) were dissected in a 6 cm Petri-dish with DPBS (Sigma) on ice. And 2 male mice were included for each suspension experiment. The following DRG single cell suspension was performed according to our previous protocol with modifications (5). Briefly, dissected DRGs were chopped with microscissors 1-2 times in 2mL Papain (25 unit/mL, Worthington Biochemical) and then incubated at 37 °C, with a mixture of digestion enzymes of Papain/Collagenase/Dispase (Papain, 25 unit/mL, 4 mL; DNase I, 55 unit/mL, 0.5 mL, Worthington Biochemical; and Collagenase and Dispase 20 mg/mL, 800 uL, Worthington Biochemical). The DRGs were triturated up and down 10 times of every 10 min using glass Pasteur pipettes with decreasing diameter (pre-coated with 0.5% BSA). Cell suspension was filtered through a 30 µm cell strainer (CellTrics, Sysmex) and washed with additional 1.5 mL artificial cerebrospinal fluid (ACSF,  87 mM NaCl, 2.5 mM KCl, NaH2PO4 1.25 mM, NaHCO3 26 mM, Sucrose 75 mM, Glucose 20 mM, CaCl2 0.5 mM, MgSO4 4 mM) and 0.5 mL DPBS. Cells were pelleted by centrifugation (300 g 6 min, 4 °C) and resuspended with 1.5 mL cold ACSF with 0.5 mL DPBS. The cell suspension was carefully loaded on top of the same volume of OptiPrep density gradient medium (Sigma) and centrifuged with 700 g ×10 min at 4 °C. The cell pellet was resuspended with 3 mL cold ACSF. SYTOX Blue (Invitrogen, ThermoFisher Scientific) was added to stain dead cells. Thereafter, alive SYTOX Blue-negative cells were sorted with fluorescence activated cell sorting (FACS, BD FACSAria Fusion / BD FACSAria III) at 4 °C. Cells were concentrated by centrifugation (300 g × 5 min, 4 °C) and resuspended with proper volume (~1 000 cells/ µL) of ACSF solution. Sorted cells were loaded onto the 10× Chromium chip G to yield single cell droplet with v3.1 kit (10× genomics). Targeted cell recovery for each well was settled to 5 000 cells (10% neurons). Reverse transcription, cDNA amplification and library construction were performed according to the user guide provided by 10× genomics.