organism: Homo sapiens
description: Protocols: In vitro organoids were dissociated for scRNA-seq based on previously published protocol (Miller et al., 2020). Firstly, all the tubes and pipette tips were coated with 1% BSA in HBSS to prevent the adhesion of cells. Next, organoids were mechanically dislodged, pipetted up and down to get rid of excess matrigel, and pooled in a Petri dish. An excess amount of media was removed, and the organoids were minced into smaller fragments in HBSS. Tissue pieces were transferred to 5mL conical tube Mix1 from Neural Tissue Dissociation Kit (Miltenyi Biotec, 130-092-628) and incubated on a shaker for 15 minutes at room temperature. Next, mix2 was added to the digestion, and every 10 minutes, the mix was agitated with a P1000 pipette tip 3-4 times. Next, cells were filtered by a 70um strainer and washed three times at 400g for 5min with 1% BSA in HBSS. Cells were counted with a hemocytometer and immediately continued with 10x Chromium for single-cell droplet generation. The use of human embryonic stem cell (ESC) line-H9 and generation of intestinal organoids were approved by the Ethics Committee of Northwest and Central Switzerland (2019-01016) and the Swiss Federal Office of Public Health. hESC line (H9) was cultured with mTESR-plus (STEMCELL Technologies) media on matrigel-coated plates (Corning, 354277). The cells were passaged when they reached 80-90% confluency every 4-5 days. hESCs were differentiated into HIOs following the previously described protocols (Capeling et al., 2020; Spence et al., 2011; Tsai et al., 2016). In brief, hESCs were patterned into definitive endoderm by Activin A (100 ng/ml) induction in RPMI-1640 media for three days with increasing concentrations of FBS (0%, 0.2%, and 2%). Then, midgut/hindgut patterning of endoderm was induced by introducing FGF4 (500 ng/ml) and CHIR99021 (2uM) with daily media changes for up to 6 days in the presence of 2% fetal bovine serum (FBS). Spheroids were collected after 96, 120, and 144 hours of patterning and embedded in matrigel (Corning, 354234), cultured in ENR media (mini gut basal media supplemented with EGF, R&D Systems, 100 ng/ml), Noggin (Peptrotech, 100 ng/ml) and R-Spondin-1-Fc (5% conditioned media)(Ootani et al., 2009). Organoid media was changed every 3-4 days. HIOs were split once the mesenchyme outgrew matrigel, and excessive cell debris accumulated in the core every 7-10 days. The mini gut basal media consists of DMEM/F-12, HEPES (Thermo Fisher Scientific, 11330032), and 1x B27 supplement (Thermo Scientific, 12587001). All media used in the differentiation process contain 1x Penicillin-Streptomycin (Thermo Scientific, 15140122). Cold dispase achieves epithelium isolation from D28 HIOs (STEMCELL Technologies, 5U/ml), based on previously reported protocol with minor modifications (Capeling et al., 2020; Tsai et al., 2018). Briefly, one full 24-well plate of HIOs was recovered from Matrigel by pipetting and transferred into a Petri dish. Then, excess matrigel and media were removed and 3 ml ice-cold dispase was added on HIOs. Organoids are incubated for 30 minutes on ice, then the dispase is replaced by 3 ml 100% FBS and incubated for 15 minutes on ice. Next, 3 ml DMEM-F12 mixed was added on organoids and pipetted up and down for a mechanical separation of epithelial and mesenchymal layers. Then, organoids were transferred to a 15 ml Falcon tube filled with DMEM media and three washes were performed at 400g for 5 minutes. While aspirating the media, mesenchymal content, which appears as a cloud on top of the pelleted epithelium, should be removed with the residual matrigel. Alternatively, epithelial pieces could be picked up individually under a stereomicroscope. We choose to use the pellet to include singlet, doublet, and multiplets and increase the chance of enteroid recovery from ISCs. Next, the pellet should be triturated 50 times in the residual volume of media and then mixed with matrigel by calculating 50ul per single well of a 24 well plate to have 9 to 12 wells of epithelium cultured in complete enteroid media (Tsai et al., 2018). After 10-14 days of culture, epithelial cysts were passaged and continued culturing organoids derived from the same pool of cysts, with or without NRG1 (R&D Systems, 100 ng/ml) in the complete enteroid medium (WENR). Next, organoid growth was monitored and passaged every 7-8 days. Complete enteroid media components consists of DMEM/F-12, HEPES (Thermo Fisher Scientific, 11330032), N-2 supplement (1x), B27 supplement (1x), penicillin/streptomycin (100U/ml), N-Acetyl-L-cysteine(2mM), Nicotinamide (20mM), EGF (100 ng/ml) and 50% conditioned media containing Wnt3a, Rspondin3 and Noggin at 1:1:1 ratio. For the first three days of enteroid culture, enteroid complete media should contain Thiazovivin (2.5μM), SB431542 (100nM), Y-27632 (10uM), and CHIR99021(4μM). Immediately after single cell dissociation, nucleic acid extraction and library preparation were performed using the Next GEM Single Cell 3' v3.1 kit according to manufacturer instructions. The libraries were prepared using the Next GEM Single Cell 3' v3.1 kit according to manufacturer instructions.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-12-31
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-12-31
attributes.tag: External Id
attributes.value: SAMEA9772124
attributes.tag: INSDC center alias
attributes.value: Institute for Translational Bioengineering
attributes.tag: INSDC center name
attributes.value: Institute for Translational Bioengineering
attributes.tag: INSDC first public
attributes.value: 2022-12-31T00:57:02Z
attributes.tag: INSDC last update
attributes.value: 2022-12-31T00:57:02Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10876:WENR
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: H9
attributes.tag: cell_type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: interstitial organoid disassociated and recultured in enteroid growth conditions
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: inner cell mas
attributes.tag: progenitor cell type
attributes.value: embryonic stem cell
attributes.tag: sample name
attributes.value: E-MTAB-10876:WENR