GSM5333164: DRG_rep9_scRNA-seq; Mus musculus; RNA-Seq
Multiple sympathetic (SGC) or sensory (DRG) ganglia were pooled together for each round of Drop-Seq. Each SCG library was generated from 4 SCGs collected from two animals, and each DRG library was generated from 6 lower lumbar DRGs collected from two animals. In total, we generated 5 SCG libraries and 11 DRG libraries. SCGs and DRGs were not collected from the same animal because of time considerations related to ganglia isolation and dissociation. Ganglia were incubated in 50units Papain diluted in HBSS plus HEPES for 20 min at 37°C then washed with HBSS plus HEPES and incubated for an additional 20 min at 37°C with 1.5mg/mL collagenase in HBSS plus HEPES. Following the second incubation, ganglia were washed again and then suspended and triturated in DMEM AIR (DMEM F12 supplemented with 12.5mM glucose and 1U/ml penicillin/streptomycin). Cells were centrifuged and resuspended in fresh DMEM AIR. Single cell suspensions were diluted to a concentration of 100cells/ul and processed with the Drop-Seq protocol as previously described (Macosko et al., 2015) including the following modifications. Flow rates for cells, beads, and oil were optimized for aquapel-treated PDMS devices purchased from FlowJem (cells and beads: 2,300ul/hour, oil: 13,000ul/hour). Up to two samples were processed in series, with single cell suspensions and stable emulsions held on ice until all collections were completed (no more than 1 hour) before proceeding immediately with reverse transcription. cDNA amplification was performed using 4000 beads/reaction with a total of 15 cycles of PCR. Sequencing libraries were prepared from amplified cDNA using the Illumina Nextera XT library prep kit