GSM9643849: day 4 hPSC-derived vein endothelial cells (unsorted) scrnaseq; Homo sapiens; RNA-Seq
Cells were collected in papain and centrifuged at 300rcf for 5 min in DNA LoBind tubes. Cell pellets were resuspended in PBS containing 0.01% BSA and passed through a 40 µm cell strainer prior to counting. Cell concentration was adjusted as needed to approximately 1,000 cells/µL, followed by a second centrifugation and repeat cell count/viability assessment. Cell suspensions were maintained on ice until loading onto the Chromium Controller. Library was prepared according to the manufacturer's instructions for the Chromium Single Cell 3'GEM Library and Gel Bead kit v3 (PN-1000075), Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (Dual Index, PN-1000121)) workflow (10x Genomics). Briefly, single cells were loaded with gel beads and partitioning oil to generate gel bead-in-emulsions (GEMs). Poly(A) RNA from lysed single cells in each GEM was reverse transcribed to barcoded cDNA containing a cell barcode and unique molecular identifier (UMI). After GEM-RT, GEMs were broken, barcoded cDNA was purified and amplified by PCR, and amplified cDNA was subjected to fragmentation, end repair and A-tailing, adaptor ligation, sample index PCR, and SPRI bead-based cleanup/size selection to generate dual-indexed gene expression libraries for paired-end Illumina sequencing. Libraries were indexed using the 10x Genomics Dual Index Plate TT Set A.