GSM4308577: NJP ganglia, replicate 1; Mus musculus; RNA-Seq
NJP ganglia were acutely harvested from adult male C57BL/6J mice (10 animals per replicate), and dissociated (75 min, 37ºC, nutation, 1 ml dissociation solution per 10 mice). Dissociation solution is Dulbecco's Modified Eagle's Medium containing liberase (55 mg/ml, Roche 05401135001) and DNAse (0.004%, Worthington, LS002007). Cells were pelleted (3 min, 300g, 4ºC), suspended (0.2% ovomucoid, 0.004% DNAse, PBS, 200 ml), triturated with a P200 pipette tip, filtered through a 100 µm mesh cell strainer, diluted (300 ml PBS passed also through the strainer), and pelleted again (3 min, 300g, 4ºC). Cells were re-suspended (1 ml, PBS-Ca2+/Mg2+, which is PBS supplemented with calcium and magnesium) and loaded onto a Percoll gradient (Sigma-Aldrich, P1644) in a 14 ml round-bottom tube with three layers: 2 ml 55% Percoll in PBS-Ca2+/Mg2+ (bottom), 2 ml 25% Percoll in PBS-Ca2+/Mg2+ (middle) and 1 ml dissociated cells (top). After centrifugation (20 min, 800g, 4ºC), the cell-enriched fraction (~1 ml) was collected at the 25%-55% Percoll interface, diluted (4 ml PBS) and centrifuged (8 min, 800g, 4ºC). The cell pellet was re-suspended (20 µl PBS-Ca2+/Mg2+), and aliquots removed for cell counting and viability analysis. Cells (10,000 per sample) were individually encapsulated in small droplets using a 10X Genomics platform. Single-cell cDNA was prepared using the Chromium Single Cell 3′ Reagent Kit (v3 Chemistry), following the manufacturer's protocol, and sequenced on an Illumina NextSeq High Output platform. Replicates 1-3 were prepared in parallel, multiplexed and sequenced on two  Illumina NextSeq High Output flow cells. Replicate 4 was prepared separately using the same protocol, and sequenced separately as a single Illumina NextSeq High Output flow cell. whole single cell RNA-seq