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 NovaSeq S1 system (100 cycles) at the Northwest Genomics Center, Seattle, USA.