Timestamp 10x Hek293:Dox
Following the droplet generation step, the reaction was transferred to a PCR strip-tube and reverse transcription, post-reverse transcription cleanup and cDNA amplification was completed following the 10x Genomics Chromium Single Cell protocol. Following cDNA cleanup, 1-5 uL of the cDNA product was PCRed with Phusion Hot Start Flex DNA Polymerase (New England BioLabs, M0535S), using a barcoded version of SGR-176 and a version of the 10x Genomics cDNA primer that included a P5 addition, with the settings: 30s of 98C denaturation; then 15-20 cycles of 10s denaturation at 98C, 20s annealing at 63C, and then 25s extension at 72C. The rest of the cDNA product was saved for the remainder of the 10x Genomics Chromium Single Cell protocol. The PCR product was then purified with Ampure beads at a 1:1 dilution, then adjusted to a concentration of 4 nM for sequencing. The libraries were sequenced on a MiSeq with the following read structure 28 Read 1, 8 Index 1, 8 Index 2, 200 Read 2.