GSM5692771: Aortic smooth muscle cell KO rep2; Mus musculus; RNA-Seq
Aortas were harvested from WT/mTmG/ Myh11ERT2 and miR-21fl/flmTmG/Myh11ERT2 mice and digested in 1.5mg/ml collagenase A (10103578001, Roche) + 0.5mg/ml elastase (6365, Worthington) buffer supplemented with 0.1% actinomycin at 37oC for 1h with gentle shaking. Digested aortas were passed through a 70 μm Cell Strainer and stained with a live/dead viability dye eFluor 450 (Thermo Fisher Scientific). The sorted eGFP+ (SMCs) and eGFP-/tdTomato+ (non-SMCs aortic cells) cells were mixed to represent complete profile of aortic cells and then encapsulated into droplets and processed following manufacturer's specifications using 10X Genomics GemCode Technology. Equal numbers of cells per sample were loaded on a 10x Genomics Chromium controller instrument to generate single-cell Gel Beads in emulsion (GEMs) at Yale Center for Genome Analysis. Lysis and barcoded reverse transcription of polyadenylated mRNA from single cells were performed inside each GEM followed by cDNA generation using the Single Cell 3' Reagent Kits v2 (10X Genomics). Libraries were sequenced on an Illumina HiSeq 4000 as 2 × 100 paired-end reads.