GSM9309587: hMHO_7; Homo sapiens; RNA-Seq
Organoid tissue was dissociated using a Papain-based based protocol using a previously published protocol. Briefly, hMHO were cut into smaller pieces, transferred to a six-well plate and incubated for 45 mins at 37°C in 3 ml enzymatic dissociation solution (containing of 30 U ml−1 papain (Worthington Biochemical, LS003127), 1× Earle's balanced salt solution (Sigma-Aldrich, E7510), 0.46% d+-glucose, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 26 mM NaHCO3, 125 U ml−1 Deoxyribonuclease I (Worthington Biochemical, LS002007) and 6.1 mM l-cysteine (Sigma-Aldrich, C7880)). The tissue in the solution was gently swirled after 20-25 mins of initial incubation. After enzyme incubation was completed, samples were transferred to a 15 ml conical tube and centrifuged at 200xg for 1 min at RT. The supernatant was removed and the samples were washed with 1 ml of inhibitor solution (2% trypsin inhibitor, Worthington Biochemical, LS00308). The samples were then triturated in a total of 1 ml of inhibitor solution in multiple steps using 200 µl of solution and a p1000 and p200 until the tissue was fully dissociated. Once dissociation was completed, 1 ml of 4% trypsin inhibitor solution was slowly added underneath the cell suspension to generate a gradient. The solution was then centrifuged at 200xg for 5 mins, the supernatant was gently removed and the resulting cell pellet was resuspended in PBS with 0.2% BSA. The cell suspension was filtered through a 40 µm Flowmi cell strainer filter (SP Bel-Art, H13680-0040) and counted with Trypan Blue using Countess Automated Cell counter. Single cell suspension was encapsulated into GEMs using Chromium 10x platform and the 3' Single Cell Gene Expression kit v3.1 (10x Genomics, 1000123) targeting 7000 or 8000 cells per lane in a 10x microfluidic chip. Single-cell libraries were prepared using 10x single index barcodes (10x Genomics, 1000213) using the 10x library construction kit (10x Genomics, 1000190).