GSM8869178: batch3etoh-1_S18; Homo sapiens; RNA-Seq
HDCs were dissociated on day 21. We harvested HDCs following the STEMCELL Agrewell400 protocol. After rinsing HDCs with phosphate-buffered saline (Corning 21-040-CV), we dissociated them with AccuMax (STEMCELL 7921) at 37C. We gently pipetted HDCs up and down for 30s every 5 min until HDCs were completely dissociated. We then quenched the dissociation with warm E6 media. After spinning down, cells were resuspended in cold E6 media and strained them through a 40 mm strainer (Fisherbrand 22-363-547). We counted cells and measured viability using Trypan Blue and Countess II (AMQAF1000, invitrogen). We then mixed cells from each line for the same condition together in equal proportions, with one mixed suspension per condition. Each mixed suspension was counted again using hemocytometer to determine the final cell concentration. We adjusted the final concentration to 800-1,200 cells/ul for each mixed suspension. Cell suspensions remained on ice before loading to the 10X Chromium Controller. We generated scRNA-seq libraries using Chromium Next GEM Single Cell 3' HT Reagent Kits v3.1 (1000348, 10X Genomics).