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Single cell RNA sequencing analysis of the mouse colonic mesenchyme (Drop-seq)

ABSTRACT: Single cell RNA sequencing of the mouse colonic mesenchyme. Mesenchymal/lamina propria cells were isolated from the middle and distal colon of wild type mice and were pooled. The vast majority of intestinal epithelial cells were depleted by Ethylenediaminetetraacetic acid (EDTA) treatment of the tissue samples and mesenchymal/lamina propria cells were isolated after enzymatic treatment with collagenase XI and dispase. Single cell RNA sequencing was performed with the Drop-seq protocol.

ORGANISM(S): Mus musculus

PROVIDER: GSE185638 | GEO | 2021/10/14

REPOSITORIES: GEO

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Project description:Resident human lamina propria immune cells serve as powerful effectors in host defense. Molecular events associated with the initiation of an intestinal inflammatory response in these cells are largely unknown. Here, we aimed to characterize phenotypic and functional changes induced in these cells at the onset of intestinal inflammation using a human intestinal organ culture model. In this model, healthy human colonic mucosa was depleted of epithelial cells by EDTA treatment. Following loss of the epithelial layer, expression of the inflammatory mediators IL-1β, IL-6, IL-8, IL-23p19, TNF-α, CXCL2 and the surface receptors CD14, TLR2, CD86, CD54 was rapidly induced in resident lamina propria cells in situ as determined by qRT-PCR and immunohistology. Gene microarray analysis of lamina propria cells obtained by laser-capture microdissection provided an overview of global changes in gene expression occurring during the initiation of an intestinal inflammatory response in these cells. Bioinformatic analysis gave insight into signalling pathways mediating this inflammatory response. Furthermore, comparison with published microarray datasets of inflamed mucosa in vivo (ulcerative colitis) revealed a significant overlap of differentially regulated genes underlining the in vivo relevance of the organ culture model. The organ culture model characterized may be useful to study molecular mechanisms underlying the initiation of an intestinal inflammatory response in normal mucosa as well as potential alterations of this response in inflammatory bowel disease. Gut specimens were derived from individuals undergoing resection for localized colon cancer. Microscopically normal colonic mucosa was dissected from the surgical specimen near the resection margin and immediately subjected to the experimental procedures. After extensive washing the mucus layer was removed by dithiothreitol (DTT) treatment. Subsequently, punches of defined surface area were prepared and denuded of epithelial cells by exposure to EDTA. Samples were collected prior to culturing and washing (control, t = 0 h, total mucosa, TM) as well as after loss of the epithelial layer (t = 5 h, mucosa after loss of epithelial layer, LEL-M) and snap frozen in liquid nitrogen. Lamina Propria (LP) was subsequently isolated via Laser Capture Microdissection (LMD) followed by RNA isolation. Microarray analysis of TM-LP (control) and LEL-LP samples was performed using the WG-DASL Human HT_12 V4 Expression BeadChip Assay from Illumina employing a minimum of 200 ng total RNA per sample. Four replicates from individual experiments were measured for each time point.

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Single cell RNA sequencing analysis of the mouse colonic mesenchyme (Drop-seq)

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