GSM6656594
Bilateral L4-5 DRGs were collected from mice at day 7 after bilateral sciatic CCI or sham surgery. Male and female mice from the same litter were subjected to the same surgery (sham or CCI). Bilateral L4-5 DRGs were collected from each mouse and DRGs from five mice of the same group (20 DRGs in total) were pooled as one sample for sequencing. The four groups included Female-Sham, Male-Sham, Female-CCI, and Male-CCI. DRGs were dissected out, digested with 1 mg/mL type I collagenase (Thermo Fisher Scientific) and 5 mg/mL dispase II (Thermo Fisher Scientific) at 37°C for 70 min (10 DRGs/tube), and disassociated into single cells in Neurobasal medium containing 1% bovine serum albumin (BSA). Cells were filtered through a 40 µm cell strainer and centrifuged at 500×g for 5 min. The pellet was resuspended in 500 μL of Triple Express (Thermo Fisher Scientific) and digested at 37°C for 2 min. The reaction was stopped with 1 mL of fetal bovine serum. The cell suspension was laid onto 5 mL of Neurobasal medium containing 20% Percoll and centrifuged at 1,400 RPM for 8 min. The upper layer was removed first and then the lower layer. The pellet was washed with 2 mL of Neurobasal medium containing 1% BSA and centrifuged at 500×g for 5 min. The pellet was resuspended with 500 μL of Neurobasal medium containing 1% BSA. The GFP+ cells were sorted into 500 μL of Neurobasal medium containing 1% BSA and centrifuged at 500×g for 5 min to remove most supernatant. The pellet was resuspended with the remaining supernatant to a concentration of ~1000 cells/μL. The single cell suspensions were further processed with Chromium Next GEM Single Cell 3′ GEM, Library & Gel Bead Kit v3 (PN-1000094) according to the manufacturer's instructions to construct the scRNA-seq library.