scRNAseq of Mouse Ascending Aorta: Mef2c-Cre YFP
Male mice of 14 weeks were anesthetized with ketamine and xylazine. After exsanguination with phosphate-buffered saline via cardiac puncture, aortas connected to the heart were excised, and the proximal aorta was isolated. The tissues were washed in ice-cold phosphate-buffered saline, minced, and pooled from two mice. The pooled tissues were digested with liberase and elastase in Hanks Balanced Salt Solution, strained through a 70 m cell strainer, and centrifuged. The cells were resuspended in 0.05% fetal bovine serum in Hanks Balanced Salt Solution. For single-cell RNA sequencing, 1 g/mL of actinomycin D was added to the phosphate-buffered saline and enzymatic cocktail. Cells were stained with DAPI for viability and sorted using a BD Aria III cell sorter. Separate single-cell libraries were created from YFP+ and YFP cells using the 10X Chromium Next GEM Single Cell 3' Kit v3.1 and sequenced on the Illumina NextSeq 550 (75 cycles).