GSM5830922
Cell dissociation protocols were optimized to achieve single cell suspensions with >90% viable cells and low number of doublets. Organoids collected at day 1, 2, 3, and 5 of our differentiation protocol were pooled in pre-warmed PBS, transferred to pre-warmed accutase and incubated for 5-7 min at 37°C. For Day 1 organoid cell suspension, 30 organoids were pooled. For Day 2 organoid cell suspension, 15 organoids were pooled. For Day 3 cell suspension, 8 organoids were pooled. For Day 5 cell suspension, 5 organoids were pooled. Organoids were briefly rinsed in PBS, then transferred to 500 µl PBS/0.05% BSA and carefully triturated to generate a single-cell suspension. Cell suspension was run through a cell strainer (Falcon, cat. no. 352235) and transferred to a 1.5 ml tube. Cells were spun down at 250g for 3 min at 4°C. Cell pellet was resuspended in 25 µl PBS/0.05% BSA, cell concentration and viability was measured using an automated cell counter, and cell suspension was further diluted as appropriate to reach the optimal range for 10x (700-1200 cells per µl). Cells were subjected to single-cell RNA sequencing (10x Genomics, Chromium Single Cell 3' v3) aiming for the following target cell numbers: Day 1, 3,000 cells; Day 2, 4,000 cells; Day 3, 5,000 cells; Day 5, 6,000 cells. Estimated actual cell numbers collected were: Day 1, 2,930 cells; Day 2, 4,977; Day 3, 5,968 cells; Day 5, 4,841 cells. Single-cell libraries were generated using standard protocols. Libraries were sequenced together on a NovaSeq 6000 system resulting in 800 million reads.