GSM5414125: AP8; Homo sapiens; RNA-Seq
For 10X single-cell RNAseq, the cells were dissociated by incubating with 0.25% trypsin for 10min at 37°C, and then an equal volume of 0.1mg/ml collagenase IV (ThermoFisher Scientific) was added for further 20min incubation at 37°C. During this incubation, the cells were triturated by pipetting with 200ml tip every 10min. The cells were resuspended in DMEM/F12 supplemented with 0.1% BSA, washed twice and then filtered through 30mm mesh. 16,000 cells were resuspended in 47ml DMEM/F12 supplemented with 0.04% BSA for further processing. Single-cell RNA-seq libraries were prepared in the Cancer Research UK Cambridge Institute Genomics Core Facility using the following: Chromium Single Cell 3′ Library & Gel Bead Kit v3 (10x Genomics, PN-1000075), Chromium Chip B Kit (10x Genomics, PN-1000073) and Chromium Single Cell 3' Reagent Kits v3 User Guide (Manual Part CG000183 Rev C, 10X Genomics). Cell suspensions were loaded on the Chromium instrument with the expectation of collecting gel-beads emulsions containing single cells. RNA from the barcoded cells for each sample was subsequently reverse-transcribed in a C1000 Touch Thermal cycler (Bio-Rad) and all subsequent steps to generate single-cell libraries were performed according to the manufacturer's protocol with no modifications. cDNA quality and quantity were assessed with Agilent TapeStation 4200 (High Sensitivity 5000 ScreenTape) after which 25% of material was used for gene expression library preparation.