GSM7506304: SST_FixedCells_scRNAseq_P5; Mus musculus; RNA-Seq
The neocortex of P5SstCre/+;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. 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 and the Fixable Viability Dye eFluor 780 1:1000 was added for 5 minutes. Cells were then transferred to 1.5 ml LoBind tubes precoated with 3% BSA overnight and centrifuged at 400g for 7 minutes. The Fixable Viability Dye was removed, pellet cells were resuspended with Fixation buffer (check protocol below for detailed information) overnight at 4°C. The day after the cells were centrifuged at 850g for 7 minutes and resuspended in 700 uL of DPBS with 1% BSA, tissue chunks were eliminated by passing cells suspension through a 40 μm cell strainer (BD). For Probe Hybridization, GEM barcoding, GEM recovery and pre-amplification, and library construction we followed the Chromium Fixed RNA Profiling protocol (Chromium Fixed RNA Profiling kit; CG000477|Rev C).