10X-single cell seq of B cells during colitis (d0 and d14)
Ontology highlight
ABSTRACT: B cells expand during the recovery after DSS-induced colonic inflammation. To investigate if a specific subtype is expanding and to analyse the transcriptional profile of these cells a transcriptional analysis on single cell level was carried out. In detail, B cells were isolated from the colon of mice on day 0 (no treatment) or day 14 (recovery) after 7 days of DSS treatment by flow cytometric sorting and analyzed by 10X sequencing.
Project description:B cells expand during the recovery after DSS-induced colonic inflammation and might play a role in influencing tissue repair. To analyze the impact B cells might have on intestinal epithelial cells and stromal cells during recovery after intestinal injury the transcriptional profile of these mice was analysed in mice depleted of B cells and control mice on day 14 after DSS colitis.
Project description:Mice were exposed to 3% DSS in the drinking water for 7 days followed by 3 days of recovery. Colon tissues were collected at 3 day after recovery in RNA later and RNA was extracted using DNA, RNA, protein purification kit from Macherey-Nagel.
Project description:Background & Aims: Dextran sulphate sodium (DSS) induced colitis in rats is one of the most widely used models of inflammatory bowel disease. Animal models can provide new insights into the pathogenesis of intestinal inflammation, which is still unknown. We have performed a genomic analysis of the DSS rat colitis including an acute and a recovery phase. Methods: Expression profile of 6 control rats were compared with colitic rats at day 1 every other day until day 23 after DSS treatment using the GeneChip Rat Genome 230 2.0 Array (Affymetrix). Functional and pathways analysis were made with the differentially expressed genes. Keywords: Time course and differentially expressed genes analysis
Project description:Background & Aims: Dextran sulphate sodium (DSS) induced colitis in rats is one of the most widely used models of inflammatory bowel disease. Animal models can provide new insights into the pathogenesis of intestinal inflammation, which is still unknown. We have performed a genomic analysis of the DSS rat colitis including an acute and a recovery phase. Methods: Expression profile of 6 control rats were compared with colitic rats at day 1 every other day until day 23 after DSS treatment using the GeneChip Rat Genome 230 2.0 Array (Affymetrix). Functional and pathways analysis were made with the differentially expressed genes. Experiment Overall Design: Experimental design: DSS was administered to animals in drinking water as follows:5%DSS from day 1 to 7, 3%DSS from day 8 to 15, 0% DSS from day 16 to 23. Samples were recovered at days 1, 3, 5, 7 (5%DSS), 9, 11, 13, 15 (2%DSS), 17, 19, 21 and 23 (0%DSS). According to inflammatory markers (Myeloperoxidase activity activity, body weight loss, colonic weigth/length ratio), three replicates at each time point were selected for genomic analysis and 6 control healthy rats (42 arrays). Experiment Overall Design: RNA was extracted from homogenized full-thickness colonic tissues in Trizol® reagent (Invitrogen) and purified with RNeasy affinity columns (Qiagen), according to manufacturer´s protocol. The microarray analysis was performed by Progenika Biopharma (Bilbao, Spain) on GeneChip® Rat Genome 230 2.0 Array (Affymetrix). All sample labeling (biotin), hybridization, staining and scanning procedures were carried out using Affimetrix, standard protocols (www.affymetrix.com). Normalization was carried out using Bioconductor sofware (affyPLM package).
Project description:It is crucial to decipher the host-microbiota interactions as they are involved in intestinal homeostasis and diseases. Caspase Recruitment Domain 9 (Card9) is an inflammatory bowel disease (IBD) susceptibility gene coding for an adapter protein for innate immunity toward many microorganisms. Card9 mediates colitis recovery via interleukin 22 pathway activation and Card9-/- mice have enhanced susceptibility to colitis. To reveal the mechanisms responsible of this defect in Card9-/-mice, we compared colon transcriptomics in WT and Card9-/- mice before and during DSS-induced colitis. Mice transcriptomes clusterized according to the genotype supporting a pattern clearly different in WT and Card9-/- mice. The number of up-regulated genes at day 7 was largely higher in Card9-/- compared to WT mice. Pathway analyses of the induced transcripts showed a dominance of immune-related pathway with a stronger signal in Card9-/- mice. Interestingly, NOD-like receptor signaling pathway, in which CARD9 is involved, was an exception with weaker activation in Card9-/- than in WT mice. During the recovery period at day 12, pathways involved in cell proliferation and replication were significantly activated in WT compared to Card9-/- mice confirming the healing defect in Card9-/- mice. For the induction of colitis, mice were given drinking water supplemented with 2% (w/v) Dextran sulphate sodium (DSS) for 7 days, then allowed to recover by drinking water alone for 5 additional days. 3 mice of each groups (WT and Card9-/-) were sacrified before DSS administration. 5 WT mice and 4 Card9-/- mice were sacrified 7 days after DSS administration and 5 mice of each group were sacrified at day 12.
Project description:Mice were exposed to 2.5% DSS (MW 36,000–50,000, MP Biomedicals) prepared in autoclaved drinking water for five days, after which they were provided DSS-free drinking water for 13 days. On day 12, 16.7% of mice (20 of 120) whose body weight returned to the same level as on day 1 (the change of body weight was less than 0.5 g) and exhibited an DAI of 0 or 1.00, were used as “well-recovered” mice. To identify which fecal miRNAs were affected during DSS-induced colitis and the recovery phase, we performed small RNA sequencing in feces obtained from well-recovered mice on days 0, 5, and 12 of the treatment regimen.
Project description:Purpose : The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) of colon samples of intestinal epithelial cell specific Axin1 Knockout mice and WT controls that were submitted to DSS-induced colitis and AOM/DSS-induced colorectal carcinogenesis. Methods : DSS-induced colitis was performed on Axin1flfl (WT) and Vil CreERT2;Axin1fl/fl (Axin1KOΔIEC) mice by giving 3% DSS dissolved in drinking water for 7 days and subsequently placed on regular water for recovery before sacrifice at Day 7 and D13. Methods : AOM/DSS-induced colorectal tumorigenesis was performed on Axin1flfl (WT) and Vil CreERT2;Axin1fl/fl (Axin1KOΔIEC) mice that were sacrificed at day 100 post-AOM injection to collect colorectal tumors. Methods : Colonic mRNA profiles of WT and Axin1KOΔIEC mice were generated by deep sequencing using Illumina NextSeq 500 instrument (150base-lengths read V2 chemistry in a paired-end mode)
Project description:The limitation of single-cell or bulk transcriptomic profiling is the lack of spatial topographical context. Spatial transcriptomics (ST) allows sequencing of polyadenylated transcripts from a tissue section which can be spatially mapped onto the histological brightfield image using an array of barcoded oligo-dT capturing probes. Using the 10X Visium platform, here, we unbiasedly characterized the spatial transcriptomic landscape of murine colon during mucosal healing upon dextran sodium sulfate (DSS) induced injury in mice in which B cell has been depleted and control.
Project description:Ribo-zero RNAseq experiments were carried out with mouse distal colon DSS Colitis model of genetically engineered mice. For each mouse line for: Gpsm3 (3% DSS), Aif1 (3% DSS), Nckap1l (Hem1), Dock2 (3% DSS), Dok3 (3.5% DSS) DSS treatment for five days followed by water for 5 days, (7 days for Nckap1l). Tissue was collected at Day 10 (Day 12 for Nckap1l (HEM1)).