Animals and Cell Lines
Animal Work
Mouse strains C57BL/6J and Cdx2:CreER; APCfl/fl; KrasWT were sourced from The Jackson Laboratory. The mice were kept in individually ventilated cages under controlled temperature and humidity, allowing a maximum of five per cage with unrestricted access to food and water in a pathogen-free environment certified by the relevant authorities. The cages were furnished with specific bedding, a nestlet, and a mouse hut. A light-dark cycle of 12 hours was maintained. All procedures involving the animals received prior approval from the Institutional Animal Care and Use Committee at Harvard University. Euthanasia criteria were established, including significant weight loss, the presence of bloody stools for a certain period, or severe lethargy, which were not exceeded during the experiments.
Cell Culture
Human embryonic kidney 293T (HEK293T) cells were cultured in DMEM supplemented with fetal bovine serum and penicillin-streptomycin. The incubator was maintained at 37°C with 5% CO₂, and cells were kept in the exponential growth phase.
Mouse Organoid Derivation and Culture
Colitis organoids were derived from colonic tissue after a specified period following DSS treatment. The animals were anesthetized before tissue processing to eliminate peripheral immune cells. The epithelial cells were extracted using a specific dissociation protocol, washed, and mixed with a gel matrix. The organoids were cultured in a specialized media and passaged regularly. They underwent treatments to minimize cell contamination before analysis.
Human Organoid Derivation and Culture
Human organoid lines were established from biopsies from patients undergoing endoscopic procedures, with necessary consent obtained. These were cultured and maintained using growth media specifically formulated for organoid expansion and differentiation. Media changes occurred regularly during the culture period.
Experimental Procedures
Colitis Induction
Mice aged 8-15 weeks were treated with dextran sulfate sodium dissolved in water to induce chronic colitis. Their weight was monitored daily, and assessments for blood in stool were conducted at intervals to gauge disease severity. Adjustments to the DSS concentration were made as needed, with specific time points designated for various stages of injury and recovery.
Colon Tissue Processing and Cell Sorting
For colitis memory studies, the colons were prepared and dissociated into single-cell suspensions. The cells underwent a staining process to discriminate various cell types before being sorted into specific populations for analysis.
Histology and Colitis Scoring
The exhibit prepared tissues underwent fixation and staining processes for histological evaluation. Scoring was carried out in a blinded manner to assess immune infiltration and other metrics.
Immunofluorescence
The fixed tissue samples were processed for immunofluorescence, involving antibody staining and imaging techniques to analyze cellular characteristics.
SHARE-seq
The SHARE-seq methodology was adapted for various sample types, including cell and organoid experiments, following specific protocols to ensure accurate results were obtained.
Organoid Proliferation and AP-1 Inhibition
Organoids were treated to inhibit a specific transcription factor, followed by assays to measure proliferation rates and assess cellular responses under different conditions.
Barcode Vector Cloning and Library Construction
Initial steps involved modifying the vector for effective cloning before constructing libraries for analysis.
Organoid Lentiviral Infection
Lentivirus generation and application were carried out to introduce genetic material into organoids, following a specific infection protocol that ensures efficiency and accuracy.
Data Processing and Analysis
Subsequent data processing involved various computational techniques to analyze and interpret the generated datasets. Steps were taken to ensure that the data were accurately mapped to the relevant genomic references.
Statistical Analysis
Throughout the study, repeated experiments were conducted, with specific numbers and arrangements noted for transparency in results and analyses. Statistical methods were employed to ensure the accuracy of the data interpretations.
Reporting Summary
For further insights regarding research design, additional information is available in the associated supplementary materials.
