GSM7856939: Aorta, Control, scRNAseq; Mus musculus; RNA-Seq
Aortic tissues were pre-digested with 1mg/mL Collagenase A (Roche, Cat No. 11088785103) for 7 min at 37 oC to remove the adventitia. Aortic tissues were then minced into 2-mm pieces and digested in DMEM (10% FBS) with 1.5 mg/mL Collagenase A and 0.5 mg/mL Elastase (Worthington, Cat No. LS006365) for 40 min at 37oC while shaking. The digested aortas were passed through a 40-mm filter to obtain single-cell suspensions. Total cell viability was obtained using live/dead viability dye eFluorTM 780 (Thermo Fisher Scientific, Cat No. 65-0865-14). Viable cells were sorted by a FACS Aria III (BD Biosciences) into 0.4% BSA/PBS Sorted aortic cells were encapsulated into droplets and processed following manufacturer's specification 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 (GEM) at the Yale Center for Genome Analysis. Lysis and barcoded reverse transcription of polyadenylated mRNA from single cells were performed inside each GEM followed by complementary DNA (cDNA) generation using the Single-Cell 3' Reagent Kits version 3.1 (10X Genomics). Libraries were sequenced on an Illumina Hiseq 4000 as 2 x 100 paired-end reads.