GSM5770207: vagus010419; Mus musculus; RNA-Seq
For each sample, vagal ganglia were acutely harvested from 10 age and gender-matched mice. Fibers were trimmed off with a micro scissors in ice cold Medium A (L-15 medium (Gibco, 11415064)), the ganglia were then digested in 0.25% Trypsin solution (Gibco, 25200056) for 10 min at 37C, followed by 1 mg/mL  collagenase I (MP Biomedicals, 0219510901) / 3 mg/mL Dispase II (Roche, 04942078001) HBSS solution for 30 min at 37C. Cells were centrifuged at 200 g for 5 min at 4C, washed twice with Medium B (L-15 medium containing 10% Fetal Bovine Serum (Gibco, 16000044)), then carefully triturated with 3 glass Pasteur pipettes with decreasing openings. Cells were pelleted, resuspended with 1 mL Medium B, and loaded onto a Percoll gradient (Sigma-Aldrich, P1644), constituted with 30% Percoll/HBSS solution in the upper layer, and 60% Percoll/L-15 solution in the lower layer. After centrifugation (800 g, 20 min, 4C), the enriched neurons at the interface of the two Percoll layers were gently collected and transferred into Medium B, and centrifuged at 200 g for 10 min at 4C. Cell pellet was resuspended in 10-15 L 0.04% BSA (Thermo Fisher, AM2616)/HBSS solution, immediately, 1 L cell solution was aliquoted for cell counting and viability assay with 0.4% trypan blue (Thermo Fisher, T10282). After that, the cell solution was subjected to 10x Gemonics for single cell RNA sequencing. Approximately 5,000-10,000 VSNs were loaded in each channel of the 10x Chromium microfluidic device to target for 3,000-6,000 cells as an output of one sample. Single cell libraries were prepared using the 10x Genomics Chromium Single Cell 3′ Reagent Kit v3 chemistry according to the manufacturer's instructions and sequenced using an Illumina NovaSeq S4 sequencer at 300 million reads to achieve a fine sequencing depth of 30,000-50,000 reads per cell. Single cell RNA sequencing, Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10x Genomics).