GSM4477424: mix15_repair; synthetic construct; OTHER
Hap cells were isolated, resuspended in single cell suspension, and mixed in a 1:1 ratio, and captured on the 10x genomics single cell RNA-seq platform Following GEM generation, GEMs were incubated at 37 C for 15 minutes, followed by 53 C for 45 minutes for RT. GEM emulsion was broken and DNA/RNA was isolated using the 10x genomics protocol using MyOne Silane beads. Repair products were separated from mRNA using and Ampure size selection as follows: 0.6x volume of Ampure XP beads were added to the purified DNA/RNA. Following 5 minute incubation, the supernatant was collected and an additional 1.2x volume of Ampure XP was added to supernatant. These repair products were digested with Pst1 (NEB R3140) for 1 hour at 37 C. These repair products then end repaired (NEB E6050) and A-tailed. Illumina TruSeq adapters were ligated followed by Illumina barcoded TruSeq PCR. See https://www.protocols.io/run/single-cell-analysis-of-functional-heterogeneity-i-uhyet7w for more detailed library protocol. Read 1 positions 1-16 contain the 16bp cell barcode, and positions 17-26 contain a 10bp UMI. Read 2 contains the repair product.