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Single cell transcriptome sequencing of CS11 and CS15 human yolk sacs

ABSTRACT: Single cell transcriptome sequencing of CS11 and CS15 human yolk sacs

PROVIDER: PRJNA564275 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10079361_1.fastq.gz	Fastqsanger.gz
	SRR10079361_2.fastq.gz	Fastqsanger.gz
	SRR10079362_1.fastq.gz	Fastqsanger.gz
	SRR10079362_2.fastq.gz	Fastqsanger.gz
	SRR10079363_1.fastq.gz	Fastqsanger.gz

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Single cell transcriptome sequencing of CS11 and CS15 human yolk sacs

Project description:Yolk sac is an important site for early embryonic hematopoiesis. However, our understanding of early hematopoietic development is still very limited. Single cell transcriptome sequencing provides us with a good research method. Here, we performed single cell RNA-seq analysis for Carnegie stage 11 (CS11) and Carnegie stage 15 (CS15) human yolk sacs.

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Project description:Primitive erythropoiesis in the mouse yolk sac is followed by definitive erythropoiesis resulting in adult erythrocytes. In comparison to definitive erythropoiesis little is known about the genes that control the embryonic erythroid program. The purpose of this study was to generate a profile of mouse embryonic yolk sac erythroid cells and identify novel regulatory genes differentially expressed in erythroid compared to non-erythroid (epithelial cells). The identification of these genes will contribute to a greater understanding of how the primitive erythroid program is controlled. This work will have clinical implications for treating sickle cell anemia and Î²-thalassemia. Activating genes in adult erythroid cells that increase embryonic or fetal globin gene expression may be a therapeutic approach to treat individuals with these disorders. Experiment Overall Design: Embryonic day 9.5 (E9.5) yolk sacs were dissected from the embryos of timed-pregnant FVB/N mice. These tissues were frozen in OCT media and 8-micron frozen sections were obtained. Laser capture microdissection (LCM) was used to isolate primitive erythroid precursors and epithelial cells from these E9.5 yolk sac frozen sections using 2 to 4 yolk sacs from 2 different litters per biological replicate. Paired erythroid and epithelial samples were collected from the same microscope slides. Total RNA was isolated from 4 different pairs of erythroid and epithelial samples and hybridized to Affymetrix 430 A 2.0 microarrays.

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Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes of Yolk Sacs of E16.5 Wild Type and Rsu1-/- mouse embryos

Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived yolk sac transcriptome profiling (RNA-seq) of E16.5 Rsu1-/- mouse embryos to that of the wild-type controls Methods: Yolk sac mRNA profiles of yolk sac isolated from E16.5 wild-type (WT) and ras suppressor 1 (Rsu1−/−) emdbryos were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500 platform. The sequence reads that passed quality filters were were mapped to human reference genome GRCh38 by HISAT2 v2.2.1 with default parameters. Results: Using an optimized data analysis workflow, we mapped about 40 million sequence reads per sample to the mouse genome and identified ___ transcripts in the yolk sacs of E16.5 WT and Rsu1−/− embryos with HISAT2 v2.2.1 workflow . Approximately __ % of the transcripts showed differential expression between the WT and Rsu1−/− yolk sac, with a fold change ≥2.0 and p value <0.05. Altered expression of 12 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of E16.5 Rsu1-/- yolk sac transcriptomes, which would expedite genetic network analyses and permit the dissection of complex biologic functions of Rsu1 during late embryogenesis.

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