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Next generation expression analysis of wild type and Acaca-/- murine small intestinal organoids

ABSTRACT: Next generation expression analysis of wild type and Acaca-/- murine small intestinal organoids

PROVIDER: PRJNA778707 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16854300_1.fastq.gz	Fastqsanger.gz
	SRR16854300_2.fastq.gz	Fastqsanger.gz
	SRR16854301_1.fastq.gz	Fastqsanger.gz
	SRR16854301_2.fastq.gz	Fastqsanger.gz
	SRR16854302_1.fastq.gz	Fastqsanger.gz

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Next generation expression analysis of wild type and Acaca-/- murine small intestinal organoids

Project description:Purpose: The goal of this study is to compare NGS-derived transcriptomes of wild type mouse small interstinal organoids and organoids deficient for the enzyme acetyl-coa-carboxylase (ACC) 1 (Acaca). Methods: mRNA profiles were generated from organoids at 24h and 96h upon in vitro deletion of the Acaca gene and the respective non-deleted wild type controls in triplicates. Total RNA was isolated using RNeasy Micro Kit (Qiagen) and libraries were prepared using NEBNext Single Cell/Low Input RNA Library Prep (New England BioLabs). The libraries were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S2 Reagent Kit (100 cycles, paired end run). Each FASTQ file gets a quality report generated by FASTQC tool. Before alignment to reference genome each sequence in the raw FASTQ files were trimmed on base call quality and sequencing adapter contamination using Trim Galore! wrapper tool. Reads shorter than 20 bp were removed from FASTQ file. Trimmed reads were aligned to the reference genome (GRCm38.) using open source short read aligner STAR (https://code.google.com/p/rna-star/) with settings according to log file. Results: the sequencing depth of our libraries is > 3 x107 reads per RNA sample. PCA analysis revealed a close relationship between WT and ACC1-deficient organoids at 24h, whereas samples at 96h were distinct from each other. Lack of ACC1 strongly reduced the expression of genes associated with intestinal epitelial stem cells. Moreover, GSEA revealed downregulation of pathways associated with DNA replication, cell cycle and chromosome segregation in ACC1-deficient organoids. Conclusions: Our study highlights the importance of ACC1-mediated cellular fatty acid synthesis for the maintenance of intestinal epithelial stem cells in mouse intestinal oganoids.

2021-11-10 | GSE188386 | GEO

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RNA-seq of murine small intestinal organoids treated with Ep300/Crebbp inhibitor I-CBP112 against untreated controls

Project description:Murine small intestinal organoids were cultured in the presence or absence of 3µM inhibitor I-CBP112 (PubChem CID 90488984), which targets E1A Binding Protein P300 (Ep300) and Creb-binding protein (Crebbp, Cbp). Organoids were grown in culture media containing EGF, Noggin and R-spondin (ENR), media was changed after 48h, and organoids were harvested after 96h.

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Project description:Murine small intestinal organoids were stimulated with the general α and β adrenergic receptor agonist norepinephrine and the complete potent and specific α2 adrenergic agonist UK 14,304 (both 1uM) or vehicle controls for 4 days. Afterwards, they were harvested for RNA isolation and proliferation and differentiation gene expression patterns were assessed.

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Project description:Type-3 innate lymphoid cells (ILC3) regulate homeostasis and orchestrate immunity in the intestine, mediated in part by their bi-directional communication with the intestinal epithelium. In order to define specific interactions between these two compartments, we used a reductionist co-culture system of murine epithelial small intestinal organoids (SIO) with ILC3. The transcriptomic profile of epithelial cells from these co-cultures was analysed by bulk RNAseq.

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Effect of norepinephrine and UK 14,304 treatment on murine small intestinal organoids

Project description:Effect of norepinephrine and UK 14,304 treatment on murine small intestinal organoids

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RNA-seq of mouse small intestinal organoids with an intestinal-epithelial specific deletion of LSD1 compared to wild type

Project description:To assess the role of LSD1 in mouse small intestinal epithelium, we grew small intestinal organoids in vitro from mice with an epithelial specific deletion of LSD1 (Villin-Cre+; Lsd1f/f) and from wild type (Villin-Cre-; Lsd1f/f) mice. This experiment uses a new Cre strain with 100% recombination efficiency. Similar to intestinal epithelium from mice with an intestinal epithelium specific LSD1-KO, Paneth cells are not present in LSD1-KO small intestinal organoids. We used these sequencing data to show intrinsic epithelial changes in the intestinal epithelium caused by LSD1 deletion in the absence of microbiota and surrounding in vivo cell types.

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Murine intestinal organoids resemble intestinal epithelium in their microRNA profiles

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2018-05-25 | GSE99237 | GEO

An Integrative Transcriptome – Proteome Approach Reveals a Robust Expression Pattern of Small Intestinal Epithelial Organoids from Germ-Free and Microbiota Associated Donors

Project description:Intestinal epithelial organoids established from resected gut tissue have become a widely used research tool. However, it remains unclear how environmental cues and other sources of variation before, during and after establishment impact on organoid properties. Divergent microbiota composition in separately held mouse lines has been identified as a confounding factor in murine in vivo studies. To probe the effect of donor microbiota on organoids, we analyzed the proteomes and transcriptomes of primary organoid cultures established from colonized and germ-free mice, and further compared them to their tissue of origin and to commonly used cell lines. The long-term global impact of donor microbiota on organoid expression patterns was negligible. Instead, stochastic culture-to-culture differences accounted for a moderate variability between independently established organoids. Integration of transcriptome and proteome datasets revealed an organoid-typic expression signature comprising 14 transcripts and 10 proteins that distinguished organoids across all donors from murine epithelial cell lines and fibroblasts. This signature, which closely mimicked expression patterns in the gut epithelium, included high levels of the inflammasome scaffold protein ASC in organoids which was lacking in commonly used cell lines. Mining of the transcriptome dataset uncovered similar high expression of also other inflammasome components, including Naip1-6, Nlrc4, and Caspase-1 in all organoids compared to the reference cell lines. Taken together, these results reveal a robust global expression pattern in organoids established from colonized and germ-free animals and suggest that organoids represent a more suitable culture model than immortalized cell lines for studies of intestinal epithelial inflammasomes.

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Analysis of the transcriptomic changes in murine small and large intestinal organoids after the sudden expression of BRAFV600E and/or p53R172H

Project description:BRAFV600E confers poor prognosis and a distinct molecular type of colorectal cancer, which is often associated with TP53 alterations. In order to understand how BRAFV600E and p53R172H promote tumorigenesis in the intestinal epithelium, we generated murine organoids harboring conditional BraffloxV600E and/or Trp53LSL-R172H alleles and conducted RNA sequencing analysis after sudden oncogene induction. As expected from their phenotypes, organoids expressing BRAFV600E, either singly or in combination with p53R172H, displayed strong changes in their transcriptome, while induction of mutant p53 alone had only little impact on gene expression. Our transcriptome analyses revealed there are profound differences in the transcriptomes of SI and COL organoids, in their oncogene naïve ground state and upon BRAFV600E expression. We observed many transcripts positively associated with proliferation, invasion and metastasis to be upregulated in both organoid types expressing BRAFV600E or BRAFV600E together with p53R172H. However, several of them were only differentially regulated in COL organoids, or displayed a more pronounced fold change in COL than in SI organoids. Interestingly, COL organoids more closely recapitulate human CRC patterns and p53R172H cooperates with mutant BRAFV600E in the regulation of several transcripts.

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