GSM8185692: Y6 endothelial cells; Homo sapiens; RNA-Seq
Y6-Ecs were washed with PBS and lifted with 0.05% trypsin for 3 minutes at 37 degrees. Digestion was stopped with media containing 5% FBS. Cells were centrifuged for 10 minutes at 300 xg and resuspended in growth media, and strained using a 30 um filter before submission to the Emory Genomics Core for 10x sequencing. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence and paired-end constructs with P5 and P7 sequences were added.