GSM7507548: Donor ascending 1; Homo sapiens; RNA-Seq
Fresh aortic root tissue was collected from the operating room within 30 minutes of excision and kept in cold HBSS (Sigma). Tissue was dissected to include all vascular layers and divided into four 50 mg pieces. Samples were then placed in digestion solution containing Liberase TM (10.4 U/mL working concentration, Roche) and elastase (8 U/mL, Sigma) and finely minced. Following 60 minutes of digestion at 37°C, digestates were filtered through a 70 μmol/L strainer, centrifuged and resuspended with fresh cold HBSS. Samples were sorted with FACS using Calcein green (ThermoFisher) for live/dead discrimination. Samples were immediately processed using a 10x Genomics microfluidics chip to generate barcoded Gel Bead-In Emulsions (GEMs) according to manufacturer protocols. Individual sample libraries were generated from single cell GEM captures using the Chromium 3' version 3 library prep protocol (10X Genomics). QC was performed following cDNA generation and final library construction.