{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Tobi Alegbe"],"organism":["Homo sapiens"],"software":["CellRanger, CellBender, Scrublet"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16998"],"description":["This scRNA-seq data resource contains auto-annotated anndata for gastrointestinal organoids derived from tissue biopsies. It consists of around 10,000 cells from 3 individuals. Samples were either stimulated with IFNG or controls."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - Libraries were sequenced using a HiSeq4000 sequencer or NovaSeq S4 XP sequencer with 100bp paired-end reads, targeting 50,000 reads per cell.","Library Construction - Cells from three organoid lines were pooled, and approximately 9,000 cells were targeted for single-cell RNA-seq using the 3′ 10x Genomics v3 chemistry, following the manufacturer’s instructions.","Sample Collection - Fresh or cryopreserved terminal ileum biopsies were obtained with informed consent from participants (three healthy donors and four individuals with Crohn’s disease).","Nucleic Acid Extraction - Organoids were derived as follows: biopsies were washed with chilled D-PBS containing Pen/Strep (100 U/mL), transferred to a 6-well plate with 2.5 mL DMEM/F12 (Thermo Fisher 11330-032), and incubated for 30 minutes at 37 °C, 5% CO₂. After incubation, biopsies were transferred to a 15 mL tube containing 5 mL of 2.5 mM EDTA in D-PBS and gently shaken on ice for 35 minutes on a rotator. Tissue was allowed to settle, and the supernatant was discarded. The tissue was washed with D-PBS, allowed to settle again, and the supernatant discarded. D-PBS was added and the tissue was pipetted up and down with a 1% BSA-coated P1000 tip to further fragment the tissue; pieces were allowed to settle and most of the supernatant was removed. Next, 2 mL of 1% BSA in DMEM/F12 was added and the suspension was pipetted 15 times with a 1% BSA-coated tip to release intestinal crypts. Tissue pieces were allowed to settle and the supernatant was filtered through a 70 µm strainer into a 1% BSA–D-PBS–coated tube. This step was repeated once and the filter was washed with fresh 1% BSA–DMEM/F12. The filtrate was centrifuged at 800 × g for 5 minutes and the supernatant was gently removed. Matrigel (Corning 356231) was prepared and mixed with released crypts by gently pipetting 10 times. The mixture was split into 2–6 domes per biopsy and plated 1 dome per well on a pre-warmed 48-well plate and incubated at 37 °C until the domes solidified. Two hundred fifty microliters of complete crypt culture medium was then added to each well (IntestiCult™ Organoid Growth Medium (Human), basal + supplement; OGM basal: 100-0190; supplement: 100-019; 1% penicillin–streptomycin (Gibco 15140122), and 10 μM final concentration of Rho/ROCK pathway inhibitor Y-27632 dihydrochloride (Stemcell 72307). Once established, organoids were passaged every 2 days from passage 1 to passage 7, depending on the sample, following this protocol. Medium was removed and, using a P1000 wide-bore tip, 250 µL cold Cell Recovery Solution (Corning 354253) was added per well. Domes were scraped and transferred to a 50 mL tube, then pipetted up and down 5 times to break Matrigel pieces. An additional 250 µL cold Cell Recovery Solution was used to wash each well, gently pipetting with a wide-bore tip to retrieve remaining organoids. The suspension was incubated on ice for 18 minutes with occasional flicking, then spun at 300 × g for 5 minutes to remove residual Matrigel. The supernatant was removed, 4 mL chilled 1% BSA in DMEM/F12 was added, and the sample was spun at 300 × g for 3 minutes at 4 °C. After removing the supernatant, Matrigel was added at a ratio of 20 µL per well of a 48-well plate. The organoid/Matrigel mixture was pipetted up and down 10 times to mix without introducing bubbles and 20 µL domes were plated into a 48-well plate. Plates were incubated at 37 °C for at least 10 minutes to solidify the Matrigel, then 250 µL complete crypt culture medium was added. Medium was changed every 2 days, and passaging was performed every 7–10 days.   Selected organoid lines were differentiated, and two cultures per line were maintained to compare unstimulated versus stimulated conditions by adding human IFN-γ recombinant protein (10 µg/mL, PeproTech®, 300-02-100UG) to the culture medium for five days to mimic chronic inflammation. Organoids were then collected and processed for single-cell dissociation as follows. Organoids from three wells per condition were collected. Two hundred microliters of Cell Recovery Solution was added to each well, Matrigel was scraped using a wide-bore tip, and the mixture was pooled into a 1.5 mL tube on ice. Tubes were kept on ice for 18 minutes, inverted every 2 minutes, then centrifuged at 300 × g for 3 minutes at 4 °C to pellet cells; supernatant was removed. Residual Matrigel was washed off twice by adding 500 µL wash buffer (HBSS −/− + 1% BSA + 2 mM EDTA) and centrifuging at 350 × g for 4 minutes at 4 °C. The pellet was resuspended in residual buffer by flicking the tube, then 500 µL pre-warmed TrypLE enzyme (Gibco 12563011) was added. Using a pre-wetted P1000 tip, the suspension was pipette-mixed to break up organoids. Tubes were incubated at 37 °C for 15 minutes until dissociation was complete, after which 500 µL cold wash buffer was added and samples were centrifuged at 300 × g for 3 minutes at 4 °C to pellet cells. ACK lysis was then performed by adding 500 µL ACK lysis buffer (Thermo Fisher A1049201), incubating at room temperature for 3 minutes, topping up with 500 µL wash buffer, and centrifuging at 300 × g for 3 minutes at 4 °C. The supernatant was poured off and cells were resuspended in residual buffer, topped up with wash buffer, and centrifuged again at 300 × g for 3 minutes at 4 °C. This wash was repeated once more, followed by a final top-up with HBSS −/− + 1% BSA and centrifugation at 300 × g for 3 minutes at 4 °C. After the final spin, the supernatant was removed, the pellet was resuspended in D-PBS with a pre-wetted P200 tip, and the suspension was filtered through a 40 µm strainer. Cells were counted under a microscope using Trypan Blue to assess viability and concentration."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - CellRanger v7.2.0 was used to demultiplex reads, align reads to GRCh38 with Ensembl version 93 transcript definitions (GRCh38-3.0.0 reference file distributed by 10X Genomics), and generate cell by gene count matrices. CellBender v2.1 55 was then applied to identify droplets containing cells and adjust the raw counts matrix for background ambient transcript contamination. Multiplets were identified and removed using scrublet v0.2.1 57, simulating 100,000 multiplets and calculating the multiplet threshold using the threshold_li function from the scikit-image package v0.17.2 58, initialised using the threshold_otsu function."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["N/A","Illumina HiSeq 4000","10X Genomics Chromium"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"pubmed_authors":["Carl Anderson","Tobi Alegbe","Tim Raine","Cristina Cotobal Martin"],"additional_accession":[]},"is_claimable":false,"name":"IBDverse: Gastroinstestinal-derived organoids","description":"This scRNA-seq data resource contains auto-annotated anndata for gastrointestinal organoids derived from tissue biopsies. It consists of around 10,000 cells from 3 individuals. Samples were either stimulated with IFNG or controls.","dates":{"release":"2026-04-28T00:00:00Z","modification":"2026-04-30T16:26:34.978Z","creation":"2026-04-28T12:47:39.945Z"},"accession":"E-MTAB-16998","cross_references":{"Biostudies":["S-BSST2944","E-MTAB-16999"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0005518","EFO_0003816","EFO_0004184"]}}