snATACseq 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 four 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. Cells were stained with 7-AAD for viability and sorted using a BD Aria III cell sorter. Nuclei were isolated from YFP+ sorted cells by incubation in a buffer containing 0.1% Digitonin, 0.1% NP-40, 0.1% Tween-20, 10mM Tris pH 7.4, 10mM NaCl, 3mM MgCl2, and 1% Bovine Serum Albumin. Libraries were prepared using the 10X Chromium Next GEM Single Cell ATAC Reagent Kit v2 according to the manufacturers protocol, involving nuclei tagmentation, barcoding, PCR amplification, and size selection of DNA fragments. The prepared libraries were sequenced on the Illumina NextSeq 2000 system at the Trapnell Lab, University of Washington.