Illumina NovaSeq 6000 sequencing; scRNA-seq of pluripotent stem cell derived human intestinal organoids
scRNA-seq of pluripotent stem cell derived human intestinal organoids
To dissociate human intestinal organoids to single cells, organoids were mechanically isolated from Matrigel droplets and then tissue minced into small fragments using a scalpel 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 37ºC incubator 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 37ºC with Mix 1. Mix 2 was added to the digestion, and tissue was incubated for 10 minute increments at 37ºC 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 4ºC and resuspended in 500µl 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/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 5,000-10,000 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 5,000 cells. Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, 16 rxns PN-1000075 was used. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 5,000 cells. Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3, 16 rxns PN-1000075 was used.