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Mesendoderm differentiation of WT and PHD2 knockout mESCs (scRNA-seq)

ABSTRACT: mesendoderm differentiation of WT and PHD2 knockout mESCs (scRNA-seq)

PROVIDER: PRJNA1028512 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR26394382_1.fastq.gz	Fastqsanger.gz
	SRR26394382_2.fastq.gz	Fastqsanger.gz
	SRR26394383_1.fastq.gz	Fastqsanger.gz
	SRR26394383_2.fastq.gz	Fastqsanger.gz
	SRR26394384_1.fastq.gz	Fastqsanger.gz

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mesendoderm differentiation of WT and PHD2 knockout mESCs (scRNA-seq)

Project description:To study the cell composition and gene expression profile in the mesendoderm differentiation of WT and PHD2 knockout, we performed scRNA-seq on the cells collected from the differentiation of WT and PHD2 knockout AB2.2 mESCs at differentiation day 4. PHD2 knockoutout mESCs were constructed by CRISPR/Cas9. The differentiation was performed using a non-lineage tendency differentiation protocol.

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Project description:PHD2 is essential in modulating HIF-1α levels upon oxygen fluctuations. Hypoxia, a hallmark of uterus, and HIF-1α have recently emerged as opposing regulators of mesendoderm specification, suggesting a role for PHD2 therein. We found that PHD2 expression initially covered the epiblast and gradually receded from the primitive streak, which was identical to hypoxia and exclusive to HIF-1α. The investigations performed in mESCs, embryoids, and mouse embryos together demonstrated that PHD2 negatively regulated mesendoderm specification. Single-cell RNA sequencing revealed that PHD2 governed the transition from epiblast to mesendoderm. The downstream effect of PHD2 relied on the HIF-1α regulated Wnt/β-catenin pathway, while it was regulated upstream by miR-429. In summary, our research highlights PHD2's essential role in mesendoderm specification and its interactions with hypoxia and HIF-1α.

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mesendoderm differentiation with Hif-1a knockdown

Project description:To study the gene expression profile in the mesendoderm differerntiation of scramble control and Hif-1a knockdown, we performed RNA-seq on the total RNA samples collected from the differentiation of scramble and Hif-1a knockdown AB2.2 mESCs at differentiation day 4. Sramble and Hif-1a knockdown AB2.2 mESCs were built by infecting AB2.2 cells with lentiviruses. Each group contained three biological replicates. The expression matrix was obtained by Hisat2 followed by Stringtie.

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Project description:The mRNA abundance in the wild-type or Phd2 knockout carotid body and adrenal medulla of mice was analysed by RNA-Seq. For this purpose, mice with tyrosine hydroxylase-restricted inactivation of Phd2 were generated. For the preparation of a single RNA-Seq library, RNA from 10 carotid bodies or adrenal medullas from 5 mice was pooled.

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Project description:To study the gene expression profile in the mesendoderm differerntiation of control and Hif-1a overexpression, we performed RNA-seq on the total RNA samples collected from the differentiation of control and Hif-1a overexpression AB2.2 mESCs at differentiation day 4.In addition, to study the gene expression profile in the mesendoderm differerntiation of Hif-1a knockdown under normoxia and hypoxia, we performed RNA-seq on the total RNA samples collected from the differentiation of Hif-1a knockdown AB2.2 mESCs under normoxia or hypoxia at differentiation day 4. Cell lines were built by infecting AB2.2 cells with lentiviruses. Each group contained three biological replicates. The expression matrix was obtained by Hisat2 followed by Stringtie.

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Project description:FGF4 stimulation increases variability in mESCs [scRNA-seq]

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Project description:Purpose: This study aimed at exploring the deregulated genes in setd2 knockout mESCs compared with wt, more particularly to find the mechanism controlled by setd2,which was required for endoderm differentiation. Methods: Setd2 wt and ko mESCs were generated by deep sequencing, using Illumina GAIIx. Using Avadis NGS (version:1.3) software to analyze the sequence reads that passed quality filter to acquire the expression level of all genes. qRT–PCR validation was performed usingSYBR Green assays. Results: Using an optimized data analysis workflow, we mapped about 80 million sequence reads per sample to the mouse genome (build mm9) and identified 17,827 transcripts in the sted2 wt and ko mESCs. About 2,516 genes were deregulated in setd2 ko mESCs, more than 10 genes were validated using qRT-PCR. Conclusions: Through RNA-seq,we noticed that a subset of genes that related to MAPK signaling pathways were down-regulated in ko mESCs. This provided a bridge to connect setd2 and mESCs endoderm differentiation. One wt and one ko mESCs were generated by deep sequencing, using Illumina GAIIx.

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