title: Illumina NovaSeq 6000 paired end sequencing; scRNA-seq of pluripotent stem cell derived human intestinal organoids grown in an alginate matrix
design: scRNA-seq of pluripotent stem cell derived human intestinal organoids grown in an alginate matrix
library_construction_protocol: 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 37C 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 37C with Mix 1. Mix 2 was added to the digestion, and tissue was incubated for 10 minute increments at 37C 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 m filter coated with 1% BSA in 1X HBSS, spun down at 500g for 5 minutes at 4C and resuspended in 500l 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/L 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 v3.1 according to manufacturer instructions. See Capeling et al., 2019 Stem Cell Reports \"Nonadhesive Alginate Hydrogels Support Growth of Pluripotent Stem Cell-Derived Intestinal Organoids\". ENR is the standard HIO media comprised of Minigut basal media, supplemented with EGF (100 ng/ml), Noggin (100 ng/ml), and R-Spondin-2 (5% conditioned media). ENR HIOs are grown in this media for their whole life span. EGF grown HIOs are grown in ENR for the first 3 days to pattern a duodenal identity, and then are switched to minigut plus EGF (no noggin or RSPO) for the rest of their life span.  Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 10,000 cells. Chromium Single Cell 3 GEM, Library & Gel Bead Kit v2, 16 rxns PN-1000075 was used. Immediately after tissue dissociation, single cell libraries were prepared on the 10x Chromium with a target of 10,000 cells. Chromium Single Cell 3 GEM, Library & Gel Bead Kit v2, 16 rxns PN-1000075 was used.