Illumina NovaSeq 6000 sequencing; scRNA-seq of pluripotent stem cell derived human intestinal organoids, adult intestine, and human fetal endoderm derived organs
scRNA-seq of pluripotent stem cell derived human intestinal organoids, adult intestine, and human fetal endoderm derived organs
TrypLE was used to dissociate hPSC-derived definitive endoderm (DE) into single cells. Briefly, excess media was removed, and digestion was carried out by incubating the DE monolayer in TrypLE (2 ml) in a 37C incubator for 10 min. Tissue was agitated using a P1000, digestion was visually assessed under a stereo microscope, and returned to the 37C incubator for another 10 min. This process continued until the monolayer was fully digested. Cells were filtered through a 70 mm filter coated with 1% BSA in 1X HBSS, spun down at 500g for 5 min at 4C and resuspended in 500ml 1X HBSS (with Mg2+, Ca2+). Cells were counted using a hemocytometer, then spun down and resuspended (if necessary) to reach a concentration of 1000 cells/mL and kept on ice. Single cell droplets were immediately prepared on the 10x Chromium according to manufacturer instructions at the University of Michigan The Advanced Genomics Core, with a target of capturing 5,000-10,000 cells. Single cell libraries were prepared using the Chromium Next GEM Single Cell 3' Library Construction Kit v2 according to manufacturer instructions. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5000 cells. Chromium Single Cell 3' Library & Gel Bead Kit v2, 16 rxns PN-120237 was used