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Expression profiling of purified cells after (mature) paneth cell depletion.

ABSTRACT: Expression profiling of purified cells after (mature) paneth cell depletion.

PROVIDER: PRJNA1185731 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR31332571.fastq.gz	Fastqsanger.gz
	SRR31332572.fastq.gz	Fastqsanger.gz
	SRR31332573.fastq.gz	Fastqsanger.gz
	SRR31332574.fastq.gz	Fastqsanger.gz
	SRR31332575.fastq.gz	Fastqsanger.gz

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Expression profiling of purified cells after (mature) paneth cell depletion.

Project description:We used transgenic mice expressing diphtheria toxin in a paneth cell specific manner to eablate paneth cells, but found that we could still detect and also purify (paneth) cells in the transgenic mice. We purified this population and a control WT population of paneth cells for expression profiling, with the goal to check if the diphtheria toxin expression system had low penetrance or if our PC population in TG mice was immature , and thus did not yet express the active diphtheria toxin.

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Project description:An indepth analysis of Paneth cell transcriptome at single cell level has not been available. Existing intestinal epithelial cell scRNA dataset contain many cell types, where Paneth cells represent a small portion. We used a flow cytometry based approach to enrich and isolate relatively pure Panethc cells from a newly developed Paneth cell reporter mouse line (Lyz1-3'UTR-IRES-CreER; Rosa26R-tdTomato). Single cell RNA sequencing was performed on purified duodenal and ileal Paneth cells of mice housed under specific pathogen free condition.

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Project description:Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their co-existence with stem cells making it difficult to culture Panth cells alone in vitro. Using a simplied toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region and surfactant assisted one pot protein digestion, we identified more than 1,300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues, a comparable proteomic coverage can be achieved with 3,600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplied workflow enables profiling of Paneth cell associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.

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