GSM9550102: Tcf21KO 16wk 3 tdt RNA; Mus musculus; RNA-Seq
Samples from mouse aorta were dissociated into single cells for RNA and ATAC sequencing using the 10x Genomics Chromium platform. Euthanized mice are perfused with phosphate buffered saline and dissected to obtain the aortic root up to the level of the brachiocephalic artery. The tissue is washed with PBS and incubated in an enzymatic dissociation cocktail containing Liberase (5401127001; Sigma-Aldrich) and Elastase (LS002279; Worthington) in Hank's Balanced Salt Solution with calcium (HBSS+ Ca2+) for 30 minutes. Mechanical dissociation is performed for 5 minutes followed by visualization under the microscope to ensure single cell suspension. This suspension is strained through a 35µm nylon mesh snap cap into falcon test tubes (352235 BD) followed by FACS sorting (Sony SH800) for tdTomato positive and negative cells in parallel. For single cell ATAC, nuclei was isolated per 10X recommended protocol and captured on the 10X scATAC platform. For both scRNA and scATAC runs, samples at some intermediate time points were processed as pooled samples with tdTomato and non-tdTomato loaded at 1:1 ratio after FACS and listed in Supplementary Data 1. Each individually sorted cell suspension was loaded onto 10x GEM G or H chips with remainder per 10x protocols (Chromium Single Cell 3' RNA V3.1, Chromium Single Cell ATAC V2). Single-cell libraries were constructed according to manufacturer's instructions with index labeling using the Dual Index Kit TT set A for scRNA-seq and Single Index kit NT for scATAC-seq. This allows for library pooling into one lane for sequencing on the Illumina NovaSeq6000 platform with a targeted depth of 40-50,000 reads per cell for RNA and 75,000 reads/cell for ATAC.