biostudies-arrayexpressXiangning DongHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15263We developed a new in vitro model of primary adult human colonic epithelium on a transwell that maintains functional barrier for up to 30 days in culture. The epithelium on the 2-dimensional transwell was derived from normal primary adult human colonoid line Colon 88 at passage 9 (3-dimensional colonoids were grown from isolated crypts of the ascending colon; University of Michigan Translational Tissue Modeling Laboratory). To determine cell composition of the model, the epithelium was collected at early (day 7), mid (day 14), and late (day 28) time points, flash frozen, and stored in liquid nitrogen for nuclei isolation followed by single nuclear RNA sequencing. Nuclei from each time point were derived from two transwells, pooled (n=2 per time point).biostudies-arrayexpressLibrary Construction - Isolated nuclei were counted and loaded into the 10x Chromium to create single-nuclei droplets targeting 5,000 nuclei. Single-cell libraries were prepared using the Next GEM Single Cell 3' Gel Bead kit v3.1 (10x genomics PN-1000147) according to the manufacturer's instructions by the University of Michigan Advanced Genomics Core.Sequencing - The University of Michigan Advanced Genomics Core's Illumina Novaseq performed all snRNA-seq.Sample Collection - Cells were gently scraped off the transwell membrane with a mini cell scraper in 200 µL of cold DPBS and transferred to a cryovial containing 1.3 mL of cold DPBS with a cut 0.1% BSA coated 200 µL pipette tip and centrifuged at 500 x g for 3 minutes at 4°C. Excess DPBS was gently removed from the cell pellet and the pellet was immediately flash frozen in liquid nitrogen. All samples were stored in liquid nitrogen until nuclei isolation.Nucleic Acid Extraction - Single nuclei were isolated from flash frozen cell pellets using Chromium Nuclei Isolation kit (10x Genomics 1000493) and protocol for Single Cell Gene Expression and Chromium Fixed RNA Profiling (10x Genomics CG000505 Rev A) with added RNase inhibitor (2 U/mL, Roche 3335402001) throughout the isolation. Because this protocol is designed for isolation of nuclei from whole tissue, we modified the initial lysis steps for gentler dissociation of the cell pellets by triturating the frozen cell pellet in cold lysis buffer 15 times with a 1 mL pipette and omitting the use of a pellet pestle. This was followed by a 5 minute incubation in the lysis buffer on wet ice with additional trituration at 2.5 minutes and 5 minutes of the incubation.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Data matrices for further analysis were generated using the Cell Ranger pipeline (10x Genomics) under standard parameters by the University of Michigan Advanced Genomics Sequencing Core.UnknownTranscriptomicsGenomicsProteomicsN/AChromium Nuclei Isolation kit (10x Genomics 1000493)Illumina NovaSeq 6000Next GEM Single Cell 3' Gel Bead kit v3.1single nucleus RNA sequencingHomo sapiensAshley CuttittaXiangning DongTristan FrumJason SpencefalseSingle nucleus RNA sequencing of an in vitro primary adult human colonic epithelium at early, mid, and late time points during long-term cultureWe developed a new in vitro model of primary adult human colonic epithelium on a transwell that maintains functional barrier for up to 30 days in culture. The epithelium on the 2-dimensional transwell was derived from normal primary adult human colonoid line Colon 88 at passage 9 (3-dimensional colonoids were grown from isolated crypts of the ascending colon; University of Michigan Translational Tissue Modeling Laboratory). To determine cell composition of the model, the epithelium was collected at early (day 7), mid (day 14), and late (day 28) time points, flash frozen, and stored in liquid nitrogen for nuclei isolation followed by single nuclear RNA sequencing. Nuclei from each time point were derived from two transwells, pooled (n=2 per time point).2025-08-01T00:00:00Z2025-06-25T14:03:19.485Z2025-06-25T14:03:19.485ZE-MTAB-15263ERP174033EFO_0002944EFO_0004170EFO_0009809EFO_0005518EFO_0003816EFO_0004184