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RNA-Seq of sorted E10.5 and E11.5 AGM cell populations from Gata3-GFP embryos

ABSTRACT: RNA-Seq of sorted E10.5 and E11.5 AGM cell populations from Gata3-GFP embryos

PROVIDER: PRJNA473139 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7218640.fastq.gz	Fastqsanger.gz
	SRR7218650.fastq.gz	Fastqsanger.gz
	SRR7218660.fastq.gz	Fastqsanger.gz
	SRR7218670.fastq.gz	Fastqsanger.gz
	SRR7218680.fastq.gz	Fastqsanger.gz

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RNA-Seq of sorted E10.5 and E11.5 AGM cell populations from Gata3-GFP embryos

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Project description:The aim of this study was to analyse the heterogeneity within the first haematopoietic stem cells generated during mouse development using single-cell RNA-Seq. Freshly dissected aorta-gonads-mesohenphros (AGM) regions from E11.5 mouse embryos were dissociated by collagenase and the cells stained for the cell surface markers EPCR and CD45. EPCR+CD45+ cells were index-sorted as single cells into 96-well plates and processed based on the SmartSeq2 protocol. PCA and tSNE analyses identified 2 main subpopulations which are segregated based on differences in cell cycle. Known HSC markers such as Gata2 and Runx1 show little correlation.

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Project description:Purpose: The goals of this study are to identify the different gene expresion level in dorsal skin cells or AGM region at different embryonic stages Methods: Dorsal skin cells were collected from different-staged embryo: E9.5, E10.5 E12.5 and E14.5. AGM were collected from different-staged embryo: E9.5, E10.5 and E11.5. In duplicate. RNA were extrated and analyzed using RNA sequencing. For dorsal skine tissues, we screened the genes were highly expressed at early embryonic stage E9.5, but not later stages (E10.5, E12.5, and E14.5). For embryonic AGM tissues, we selected the genes were highly expressed at early embryonic stage E9.5, but not later stages (E10.5 and E11.5). The gene list were filtered based on secreted potein candidates. These gene candidates might function as novel embryonic anti-tumor factors Results: Using an optimized data analysis workflow, we mapped the gene list for potential secretory anti-tumor factors. Conclusions: One of the top gene from the gene lists is NEPN which we hypotherze it has unique anti-tumor function in the early embryonic microenvirnemt and might be used as a novel cancer therapeutic drug

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ChIP-on-chip with anti-RBPj antibody from embryonic AGM E11.5

Project description:Hematopoietic Stem Cells (HSC) are originated during embryonic development from endothelial-like cells located in the ventral side of the dorsal aorta around day E10-12 of murine development. This region is called AGM for Aorta/Gonad/Mesonephros refering to the tissues around the hemogenic aorta. Hematopoiesis depends on the Notch pathway and the identification of Notch-targets is important for the understanding of blood origin. Hematopoietic Stem Cells (HSCs) specification occurs in the embryonic aorta and requires Notch activation, however which are the elements regulated by Notch that control this process are mainly unknown. Here, we took a genome-wide approach to identify putative direct Notch targets by precipitating the chromatin that binds to the Notch partner RBPj in the Aorta-Gonad-Mesonephros (AGM) tissue from E11.5 mouse embryos. This assay revealed 701 gene promoter regions as candidates to be regulated by Notch in the AGM. Chromatin was obtained from a pool of 40 dissected AGMs at E11.5. Chromatin immunoprecipitation (ChIP) was performed as previously described (Aguilera et al, PNAS 2004) with minor modifications. In brief, cross-linked chromatin was sonicated for 10 minutes, medium-power, 0.5-interval; with a Bioruptor (Diagenode) and precipitated with anti-RBPJ (Chu and Bresnick, 2004). After crosslinkage reversal, DNA was used as a template for PCR or for array hybridization. Mouse promoter chip on chip microarray SET (Agilent) was used to identify RBPj targets. It covers 70,000 best identified gene regions with a-5.5 kb to + 2.5 kb range, and has on average 25 probes per gene with an average probe to probe distance of 200 bp. The ChIP-on-chip was performed with dye swaps and one IgG control was brought along. Enrichment analysis was done by comparing the precipitation normalized dye swap signal with input control signal.

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