GSM4477420: pbmc30_repair; synthetic construct; OTHER
PBMCs were isolated from a healthy adult using standard protocols and captured on the 10x genomics single cell RNA-seq platform Following GEM generation, GEMs were incubated at 37 C for 30 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 to clean up the repair fraciton. The repair fraction was split in two to optimize the library prep. 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.