title: GSM8262313: LDS, rep2; Mus musculus; RNA-Seq
library_construction_protocol: The aorta was perfused with 5mls of PBS injected through the left ventricle of the heart. The thoracic aorta from the aortic root to the arch (sinus of Valsalva to the branchpoint of the left subclavian artery) was collected and cut into small pieces and placed into a room temperature in 500 ul of solution A (HBSS (Thermo Fisher Scientific, 14175095), 50% D-Trehalose (Quality Biosciences, A611-K480-95), RNase inhibitor [20u/ul] (Applied Biosystems, N8080119)). 1ul/ml of room temperature DyeCycle Violet (DCV) (ThermoFisher, V35003) was added to stain double stranded DNA. After all aortic samples were collected, the tissue enzymatically digested by adding 500ul of solution B (Collagenase II [315U/mg] (Worthington-Biochem, LS004176), Elastase (MilliporeSigma, E1250), Dnase I [2620U/mg] (Zymo Research, E1009) in a thermomixer set to 800 rpm for 30 minutes or 1 hour at 37oC. After incubation, each sample was gently pushed through a 3ml syringe with a 21G needle 8 times to further disrupt the tissue. 100ul of 100% FBS (Corning, 35-010-CV) was added to each sample tube to inhibit the enzymes. The tubes were spun down at 800xg for 3.5 minutes at room temperature. The supernatant was discarded, and cells were resuspended in 500ul of solution A and 1ul/ml of DCV. The cell suspension was carefully pipetted through a 40um filter cap tube. Propidium iodide (PI) (ThermoFisher Scientific, P1304MP) was added to each sample and cells were sorted by flow cytometry to produce a single cell suspension of DCV+, PI- cells. 20ul of 1% BSA in HBSS was used as catch buffer. 10x Genomics 3' single index chemistry for single cell RNA sequencing as per manufacturer's protocol. 10x Genomics Chromium 3' v3 chemistry, sequenced on an Illumina NovaSeq