GSM9286770: KPCY clone 1, 32, 33, 52, and 76 after 19 days of editing.; Mus musculus; RNA-Seq
Isolated KPCY clones (76 and 84) were quantified and combined at a ration of 1:1. Pooled cells were strained through a single 40uM cell strainer. Strained cells were pelleted via centriofuge for 300rcf for 5 minutes at RT. Supernatant was removed and the cells were resuspended in PBS+1% BSA. Cells were pelleted and the supernatant was removed again. Cells were resuspended a final time in PBS+1% BSA before being counted to determine the cell concentration. Cells were diluted to a concentration of 1,000 cells/ul and loaded into the 10X chromium sample. Library was performed according to the manufacturer's instructions (single cell 3' v4 protocol, 10X Chromium). Briefly, KPCY cells 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 each GEM was retrotranscribed into cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI), and the 10X Barcode. The pooled and barcoded cDNA was cleaned with Silane DynaBeads, amplified by PCR and the appropriate sized fragments were selected with SPRIselect reagent. Amplified and size selected cDNA molecules were fragmented and ligated with the R2 adapter sequence before indexes and P5 and P7 sequences were added by PCR.