GSM8483439: Aortic root, Q158Q; Mus musculus; RNA-Seq
The aortic root and ascending aorta (from beginning of aorta up to BCA) were isolated and transferred to an enzymatic dissociation cocktail with 4 U ml-1 Liberase (#5401127001, Sigma Aldrich), 60 U ml-1 Hyaluronidase (#H3506), and 120 U ml-1 DNase I (#D4527, all purchased from Sigma Aldrich) in Dulbecco's Modified Eagle Medium (DMEM) and slightly minced. After incubation at 37 °C for 70 min with agitation, the digestion reaction was quenched with 6 mL of HBB buffer (2% Fetal Bovine Serum (FBS) and 0.2% Bovine Serum Albumin (BSA) in Hank's Balanced Salt Solution (HBSS)), then filtered through 70 μm filters. Samples were pelleted by centrifugation at 4 °C, 1400 rpm for 5 min and the supernatant was discarded. Cells were resuspended in FACS buffer (2% FBS and 2mM Ethylenediaminetetraacetic acid (EDTA) in calcium/magnesium free PBS) and centrifugation was repeated in above conditions and supernatant aspirated. Cells were resuspended in FACS buffer with the addition of DRAQ5 (#62251, Thermo Fisher Scientific, 1:100 dilution) and incubated on ice for 30 min. Cells were washed three times with FACS buffer following same centrifugation as above and then resuspended in FACS buffer with DAPI (#564907, BD Biosciences) and filtered into filter-top FACS tubes. First singlets were gated and subsequent DRAQ5+DAPI- events were collected in 300 uL cell resuspension buffer (0.04% BSA in PBS) – collected cells were centrifuged as above and resuspended in collection buffer to a target concentration of 1,000 cells uL-1. Cells were counted on a hemocytometer before proceeding with the 10x protocol. Cells were processed using the Chromium Single Cell 5ʹ Reagent V1.1 kit from 10X Genomics.