GSM7899133: 4OH-Tam injected Kir4.1 cKO; 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 2 (V2) chemistry. cDNA and libraries were checked for quality on Agilent 4200 Tapestation, quantified by KAPA qPCR. All libraries were sequenced on individual lanes of an Illumina HiSeq4000 targeting 6,000 barcoded cells with an average sequencing depth of 50,000 reads per cell.