GSM5310090: Tcf4+/+ P12 mice; Mus musculus; RNA-Seq
Brainstem slices were prepared using a vibratome in ice cold, high sucrose slicing solution. Slicing solution was equilibrated with a 5% CO2-95% O2 gas mixture before use. Transverse brainstem slices (300 μm thick) were prepared and then immediately enzymatically treated at 34°C with protease XVIII (6 mg/mL) for 6 minutes. After enzyme incubation, slices were washed three times in cold dissociation solution and then transferred to an enzyme inhibitor mix containing trypsin inhibitor (10 mg/mL) and bovine serum albumin (BSA, 10 mg/mL) in cold sucrose dissociation solution. Shortly thereafter, slices were transferred to a glass Petri dish on ice. Using a plastic transfer pipette and scalpel, each region of interest was micro-dissected out of the slices and manually separated into sterile microcentrifuge tubes. Tissue chunks were then warmed to 34°C for 10 minutes before trituration. A single cell suspension was achieved by trituration using a 25 and 30 gauge needles sequentially, attached to a 2 mL syringe. Samples were triturated for an average of 5 minutes. Immediately after, the samples were placed back on ice and filtered through a 30-micron filter into sterile microcentrifuge tubes for cell viability assessment. Cell viability for each sample was assessed on a Countess II automated cell counter (ThermoFisher), and 12,000 cells were loaded for capture onto an individual lane of a Chromium Controller (10X Genomics). Single cell capture, barcoding and library preparation were performed using the 10X Chromium platform according to the manufacturer's protocol (#CG00052) using version 3 (V3) chemistry. cDNA and libraries were checked for quality on Agilent 4200 Tapestation, quantified by KAPA qPCR. Each library was sequenced on 16.67% of an Illumina NovaSeq 6000 S4 lane, targeting 6,000 barcoded cells with an average sequencing depth of 50,000 reads per cell.