title: HIO_control_14d
organism: Homo sapiens
description: Protocols: 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 v3.1 according to manufacturer instructions. Briefly, hPSCs were patterned into definitive endoderm (DE) by supplementing Roswell Park Memorial Institute 1640 (RPMI-1640) with Activin A (100 ng/ml) for 3 days and increasing HyClone FBS concentration (0%, 0.2%, 2%) each day of DE induction. Hindgut patterning was carried out through addition of FGF4 (500 ng/ml) and CHIR99021 (2 µm) to RMPI-1640 containing 2% HyClone FBS. Media was changed daily, and spheroids were collected after 5 days of hindgut patterning. Spheroids were embedded in Matrigel (8 mg/ml, Corning, 354234) and incubated in ENR media (Minigut basal media, supplemented with EGF (100 ng/ml), Noggin (100 ng/ml), and 5% R-Spondin-2 conditioned media for 3 days to pattern duodenal identity. After 3 days, ENR media was replaced with Minigut media containing only EGF (100 ng/ml. Minigut media is composed of the following components: Advanced DMEM:12 (Life Technologies, 12634), 1x B27 supplement (Life Technologies, 17504044), L-2 mM Glutamine (Life Technologies, 25030), 15 mM HEPES (Life Technologies, 15630080). All medias used in differentiation contain 1x PenStrep (Life Technologies, 15140). Minigut media is composed of the following components: Advanced DMEM:12 (Life Technologies, 12634), 1x B27 supplement (Life Technologies, 17504044), L-2 mM Glutamine (Life Technologies, 25030), 15 mM HEPES (Life Technologies, 15630080). All medias used in differentiation contain 1x PenStrep (Life Technologies, 15140). HIO maintenance and splitting was carried out as previously described with media changes every 3-4 days (Capeling et al., 2020; McCracken et al., 2011) 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.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-12-20T04:16:52Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-06-18T14:03:38Z
attributes.tag: External Id
attributes.value: SAMEA6990500
attributes.tag: INSDC center name
attributes.value: University of Michigan Health System - Internal Medicine - Gastroenterology
attributes.tag: INSDC first public
attributes.value: 2020-12-20T04:16:52Z
attributes.tag: INSDC last update
attributes.value: 2020-06-18T14:03:38Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9228:HIO_control_14d
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: H9
attributes.tag: cell type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: intestinal organoid culture
attributes.tag: sample name
attributes.value: E-MTAB-9228:HIO_control_14d
attributes.tag: scientific_name
attributes.value: Homo sapiens