title: GSM8441017: Female HH6 AVS, scRNAseq; Homo sapiens; RNA-Seq
library_construction_protocol: Aortic valves were excised from human hearts (post-mortem interval < 48 hours) and washed with Earle's Balanced Salt Solution (Cat. No. E2888). Briefly, valve leaflets were then digested with Liberase TM (Cat. No. 05401119001) for 30 minutes before quenching with media for centrifugation. The resulting cell pellet was treated with Red Blood Cell Lysis Buffer (Cat. No. 00433357) for 1 minute before quenching with media and centrifugation. Cells were counted manually with a hemacytometer before subsequent preparation for single cell sequencing. Library preparation was performed using manufacturer's protocols (single cell 3' v3.1). Accordingly, valve cells were resuspended in a master mix and loaded with partitioning oil and gel beads into a Next GEM chip to create gel bead-in-emulsion (GEM). Poly-A mRNA from cell lysate was reverse transcribed (RT) to generate barcoded, full length cDNA. Silane magnetic beads were used to clean cDNA from RT mixture and then cDNA was amplified via PCR for library construction. Enzymatic fragmentation and size selection with SPRIselect reagent was used to extract the optimal cDNA amplicon size. Illumina R2 primer sequences, paired end sequences P5 and P7 and a sample index were added to each sample.