{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"submitter":["Nicholas Ilott"],"instrument_platform":["Illumina HiSeq 2500"],"study_type":["RNA-seq of coding RNA"],"organism":["Mus musculus"],"species":["Mus musculus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-12414"],"description":["Intestinal organoids stimulated with conditioned media from inflamed or non-inflamed source."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Growth Protocol - Organoids were cultured as previously described.1 Colonic crypts were isolated from longitudinal opened and cleaned colon tissue of Atg16l1fl/fl mice by 2mM EDTA in PBS incubation for 90min at 4°C. The tissue was vigorously resuspended in PBS/10% FCS and the supernatant containing the crypts filtered through a 100μm cell strainer and pelleted. Crypts were seeded in 20μl Matrigel/well (Corning) in a 24-well plate. After polymerisation of the Matrigel, 400ul of complete organoid medium was added [Advanced Dulbecco's Modified Eagle Medium (DMEM)/F12 supplemented with 1% Pen/Strep, 10mM HEPES, 1X GlutaMax, 50ng/ml mEGF, 1× N2, 1× B27 (all from Invitrogen), 1mM N-acetylcysteine (NAC), 10μM RhoK-inhibitor (Y-27632 dihydrochloride monohydrate) (both from Sigma) and conditioned medium containing Wnt3a (20%), Noggin (20%) or R-spondin (10%)]. The conditioned media were generated from Rspol-Fc Hek 293T cells (kind gift from Dr. Calvin Kuo, Stanford) and Nog-Fc Hek 293T cells (kind gift from Dr. Gjis van den Brink, Amsterdam) and Wnt3A L cells (ATCC CRL-2647). The medium was changed every 2-3d and organoids were expanded over several passages before they were used for experiments with indicated stimuli. Ref: 1 Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).","Library Construction - Illumina Truseq mRNA kit","Sequencing - Illumina Hiseq2500 rapid run","Nucleic Acid Extraction - Qiagen Rneasy Kit","Sample Treatment - 10% control conditioned media for 20h","Sample Treatment - Lamina propria lymphocytes were isolated from C57BL/6 mice at 2 weeks of Helicobacter hepaticus + anti-IL10R colitis and 2.3x106 cells/ml were seeded in complete RPMI for 20h. Supernatants from cells of 7 mice were pooled and frozen at −80°C. Colonic organoids were seeded for stimulation at p4, p6 and p7. 24h prior to stimulation full medium was reduced to organoid medium containing 1% Wnt3a. Colonic organoids were stimulated with 10% control conditioned media or inflammatory conditioned media for 24h prior to lysis and RNA isolation (Qiagen RNeasy). Quadruplicates were sequenced."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","MAGE-TAB Files"],"pubmed_authors":["Kevin Maloy","Nicholas Ilott"],"additional_accession":[]},"is_claimable":false,"name":"MHC class II antigen presentation by intestinal epithelial cells fine-tunes bacteria-reactive CD4 T cell responses","description":"Intestinal organoids stimulated with conditioned media from inflamed or non-inflamed source.","dates":{"release":"2023-11-09T00:00:00Z","modification":"2022-12-01T16:03:05.816Z","creation":"2022-12-01T16:03:05.816Z"},"accession":"E-MTAB-12414","cross_references":{"ENA":["ERP143000"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003738","EFO_0004184","EFO_0003969"]}}