GSM7656796: 96 glial cells_plate4; Mus musculus; RNA-Seq
Vagal ganglia were dissected from 4 Phox2bCre;Ai14 mice at P4 of either sex and placed into ice cold Ringer's solution (0.125 M sodium chloride, 1.5 mM calcium chloride dihydrate, 5 mM potassium chloride, 0.8 mM sodium phosphate dibasic). Ganglia were transferred to a 1.5 ml Eppendorf tube with 1.5 ml HBSS without calcium, magnesium and phenol red (Thermo Fisher Scientific), 3 µl saturated NaHCO3, 1 mg L-Cysteine and 60 U Papain (Sigma-Aldrich), placed at 37°C and shaken gently for 12 minutes. The 1.5 ml Eppendorf tube was centrifuged at 300 RCF and the solution was discarded, taking care not to disturb the ganglia. A prewarmed (37°C) enzyme solution containing 1 ml HBSS without calcium, magnesium and phenol red (Thermo Fisher Scientific), 4 mg Collagenase NB4 (Serva) and 4.6 mg Dispase II (Gibco) was added to the ganglia, placed at 37°C and shaken gently for 12 minutes. The 1.5 ml Eppendorf tube was centrifuged at 300 RCF and the solution was discarded, taking care not to disturb the ganglia. Prewarmed (37°C) 0.5 ml HBSS without calcium, magnesium and phenol red (Thermo Fisher Scientific) was added to the ganglia, which were triturated with a 1 ml plastic pipette tip until the ganglia began to dissociate, and then with a 200 µl plastic pipette tip until the ganglia were completely dissociated. The dissociated ganglia were passed twice through a 40 µm filter (Falcon) and DAPI (Sigma) was added to a final concentration of 300 nM to label dead cells before sorting. Single cell RNA-sequencing was done following the CEL-Seq2 protocol (Hashimshony et al., 2016). cDNA Libraries were prepared for 4 96-well plates, pooled together, and sequenced on 1 lane of a HiSeq 1500 (Ilumina) by the next generation sequencing core facility at the Max Delbrück Center for Molecular Medicine.