Single-Cell RNA Sequencing of Kelly Cells

Kelly cells (sensitive, intermediate and resistant states) were grown to 70% confluence in T75 culture flasks. In brief, growth medium was aspirated and cells were treated with 0.25% Trypsin/EDTA for 3 min at 37 °C, after which cells were washed twice with 1× PBS. Cells were then resuspended into single cells at a concentration of 1 × 106 per ml in 1× PBS with 0.4% BSA for 10x Genomics processing. The sorted cell suspensions were loaded onto a 10x Genomics Chromium instrument to generate single-cell gel beads in emulsion (GEMs). Approximately 5,000 cells were loaded per channel. scRNA-seq libraries were prepared using the following Single Cell 3′ Reagent Kits: Chromium Single Cell 3′ Library & Gel Bead Kit v2 (PN-120237), Single Cell 3′ Chip Kit v2 (PN-120236) and i7 Multiplex Kit (PN-120262) (10x Genomics) as previously described^{42 (link)}, and following the Single Cell 3′ Reagent Kits v2 User Guide (Manual Part CG00052 Rev A). Libraries were run on an Illumina HiSeq 4000 system (SY-401–4001, Illumina) as 2 × 150 pairedend reads, one full lane per sample, for approximately >90% sequencing saturation.