GSM5599214: S1; Homo sapiens; RNA-Seq
Organoids were dissociated using Accutase, serially filtered to remove debris, and were subjected to high-throughput FACS (Aria II flow cytometer, Becton Dickson). This procedure allowed us to isolate only live cells in a defined concentration (2000 live cells/uL) that could be standardized between lines for microfluidic device loading. Cell viability was confirmed using Countess-II (Invitrogen, Thermofisher, CAT: AMQAX1000). Next, cells were prepared for 10X Genomics Chromium library preparation according to manufacturer instructions (Single-Cell 3' Library, Gel Bead and Chromium CHIP kit, 10X Genomics). Barcoded emulsions were broken, amplified and libraries prepared using the 10X Chromium kits. cDNA was examined on a Fragment Analyzer running PROSize v.3/0 3.0.1.6. Single-Cell 3'