GSM7102365: Lung tumor with bleomycin treatment CAFs; Mus musculus; RNA-Seq
Mice were euthanized by intracardiac perfusion with 15 mL of PBS-heparin under isoflurane anesthesia. Tissues were manually minced and digested in 20 mL of Hank's Balanced Salt Solution (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 2 mg/mL of collagenase A (Roche, Basel, Switzerland) and 1× DNase I (Sigma-Aldrich, St. Louis, MO, USA) for 30 min (20 min for normal tissue) at 37°C with agitation. After digestion, the cell suspensions were quenched with 5 mL of FBS and filtered through 40 μm nylon mesh. The filtered suspensions were then pelleted by centrifugation (1,800 rpm for 4 min at 4°C) and resuspended in flow cytometry buffer [PBS containing 1% bovine serum albumin (BSA) and 5 mM EDTA] as a single cell suspension. Following tissue digestion, single cell suspensions were blocked with rat anti-mouse CD16/CD32 antibodies (eBioscience, Waltham, MA, USA) for 10 min on ice, and pelleted by centrifugation. The cells were subsequently labeled with 100 µL of fluorophore-conjugated anti-mouse extracellular antibodies at recommended dilutions for 30 min on ice in flow cytometry buffer. Cells were then washed and immediately sorted for immune cells (CD45+) or mesenchymal/CAF cells (CD45-, CD31-, EpCAM-) on Aria-II (BD Biosciences). After cells were sorted into FACS buffer, the were pelleted by centrifugation, resuspended in 0.04% BSA in PBS, and cell counts were obtained. Sorted cells from each sample were submitted to McDonnell Genome Institute at WUSM, where they were encapsulated into droplets and libraries were prepared using Chromium Single Cell 3'v3 Reagent kits according to the manufacturer's protocol (10x Genomics, Pleasanton, CA, USA). The generated libraries were sequenced by a NovaSeq 6000 sequencing system (Illumina, San Diego, CA, USA) to an average of 50,000 mean reads per cell.