GSM9435411: SST_scRNAseq_E16.5 (2); Mus musculus; RNA-Seq
The neocortex of P5 SstCre/+;RCEfl/+ mice were dissected in ice-cold HBSS buffer supplement with 25 mM of glucose. Tissues were cut into small pieces and digested at 34°C with carbogen oxygenation using 20 ml of pronase dissociation buffer solution, with the following composition: 0.2 mg/ml Pronase, 25mM of Trehalose, 10 mM glucose, 0.8 mM kynurenic acid, 0.05 mM APV, 0.09M Na2SO4, 0.03M K2SO4 and 0.014M MgCl2. After 10 minutes of incubation, 500 μl 2.5mg/ml Liberase and 400 μl of 10mg/ml DNAseI were added and incubated at 34°C for 5 more minutes. Post enzymatic digestion, tissues were washed once in ice-cold dissociation buffer without enzyme and the mechanically triturated into single cells in 5 ml of OptiMEM supplement with 1% Trehalose, 10 mM glucose, 0.8 mM kynurenic acid and 0.05 mM APV using 5 ml volumetric pipette with 1 ml not filtered tip. For sample 3, cells were incubated with fixable viability dye 780 and fixed for 15min at RT with 3% Glyoxal.  We removed myelin by overlaying single cell suspension on top of a 20% percoll and centrifuge at 700 g for 10 mins. The cell pellet was resuspended in 300-500 μl of OptiMEM with 1% Trehalose, 10mM glucose, 0.8 mM kynurenic acid, 0.05 mM APV. Tissue chunks were eliminated by passing cells suspension through a 40 μm cell strainer (BD) and 1ul DAPI 100uM was added prior the sorting to sample 1 and 2. Libraries were performed according to the manufacturer's instructions (single cell 3' v3.1, 10X Genomics)