For single cell RNA sequencing, hMGEOs or hCOs were collected from different culture wells, combined, and dissociated into single cells (early stage group-1 (day 30): 8 hMGEOs and 8 hCOs; early stage group-2 (day 30): 12 hMGEOs and 10 hCOs; late stage group-1 (day 72): 7 hMGEOs and 8 hCOs; late stage group-2 (day 79): 11 hMGEOs and 10 hCOs). Cells dissociated from organoids were suspended in 1% BSA/PBS + 10 μM Y27632 and stained with propidium iodide (PI) for 15 minutes on ice. PI-negative cells were collected by FACS and re-suspended in 0.04% BSA/PBS at a concentration of 128 cells/uL to generate cDNA libraries with the Single Cell 3′ Reagent Kits, according to the manufacturer's instructions. Briefly, cells were partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs). Using microfluidics, cells were flowed at a limiting dilution into a stream of Single Cell 3′ Gel Beads and then a stream of oil. This ideally encapsulates a single cell and an individual gel bead in an oil suspension. Upon cell lysis and dissolution of the Single Cell 3′ Gel Bead within the droplet,
