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
Use of human tissue was reviewed and approved by The University of Michigan Institutional Review Board (IRB). De-identified human fetal intestine tissue was obtained from the University of Washington Laboratory of Developmental Biology. Tissue was shipped overnight in Belzer-UW Cold Storage Solution (ThermoFisher, NC0952695) with cold packs. Cell dissociations were carried out similar to previously published methods (Miller et al., 2020). To dissociate human fetal tissue to single cells, tissue was mechanically minced into small fragments, and in a petri dish filled with ice-cold 1X HBSS (with Mg2+, Ca2+). This tissue was then transferred to a 15 mL conical tube. Dissociation enzymes and reagents from the Neural Tissue Dissociation Kit (Miltenyi, cat. no. 130-092-628) were used, and all incubation steps were carried out in a refrigerated centrifuge pre-chilled to 10ºC unless otherwise stated. All tubes and pipette tips used to handle cell suspensions were pre-washed with 1% BSA in HBSS to prevent adhesion of cells to the plastic. Tissue was treated for 15 minutes at 10ºC with Mix 1. Mix 2 was added to the digestion, and tissue was incubated for 10 minute increments at 10ºC until digestion was complete. After each 10 minute incubation, tissue was agitated using a P1000, and tissue digestion was visually assessed under a stereo microscope. This process continued until the tissue was fully digested. Cells were filtered through a 70 µm filter coated with 1% BSA in 1X HBSS, spun down at 500g for 5 minutes at 10ºC and resuspended in 500µl 1X HBSS (with Mg2+, Ca2+). 1 mL Red Blood Cell Lysis buffer (Roche cat. No 11814389001) was then added to the tube and the cell mixture was placed on a rocker for 15 minutes at 4ºC. Cells were spun down (500g for 5 minutes at 10ºC), and washed twice by suspension in 2 mLs of HBSS + 1% BSA followed by centrifugation. Cells were counted using a hemocytometer, then spun down and resuspended (if necessary) to reach a concentration of 1000 cells/µL 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 cells. Single cell libraries were prepared using the Chromium Next GEM Single Cell 3' Library Construction Kit (v2 or v3) 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