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Bulk RNA-seq analysis of Etv2 induced EB differentiation.

ABSTRACT: Bulk RNA-seq analysis of Etv2 induced EB differentiation.

PROVIDER: PRJNA770422 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR16293131_1.fastq.gz	Fastqsanger.gz
	SRR16293131_2.fastq.gz	Fastqsanger.gz
	SRR16293132_1.fastq.gz	Fastqsanger.gz
	SRR16293132_2.fastq.gz	Fastqsanger.gz
	SRR16293133_1.fastq.gz	Fastqsanger.gz

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Bulk RNA-seq analysis of Etv2 induced EB differentiation.

Project description:The vasculature is an essential organ for the delivery of blood and oxygen to all tissues of the body and thus relevant to the treatment of ischemic diseases, injury-induced regeneration, and solid tumor growth. Therefore, the definition of pioneer factors and regulatory pathways that govern the specification and differentiation of endothelial progenitors will serve as a platform for targeted therapies to promote cardiovascular regeneration and treatment of solid organ tumors. Previously, we demonstrated that ETV2 was an essential transcription factor for the development of cardiac, endothelial and hematopoietic lineages. Here, we report that ETV2 functions as a novel pioneer factor that relaxes closed chromatin and regulates endothelial development. By comparing engineered embryonic stem cell differentiation and reprogramming models with single cell RNA-seq, ATAC-seq and ChIP-seq techniques, we demonstrated that ETV2 was able to bind nucleosomal DNA and function as a pioneer factor independent of the cellular context. We determined that ETV2 executes a pioneering role by recruiting BRG1 to remodel chromatin around endothelial genes and help maintain an open configuration. ETV2 chromatin binding also resulted in increased H3K27ac deposition. Collectively, these results will serve as a platform for the development of therapeutic initiatives directed towards cardiovascular and oncological diseases.

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ATAC-seq analysis of Etv2 induced MEF reprogramming and EB differentiation.

Project description:ATAC-seq analysis of Etv2 induced MEF reprogramming and EB differentiation.

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ChIP-seq analysis of Etv2 induced MEF reprogramming and EB differentiation.

Project description:ChIP-seq analysis of Etv2 induced MEF reprogramming and EB differentiation.

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ChIP-seq analysis of Etv2 induced MEF reprogramming and EB differentiation.

Project description:ChIP-seq analysis of Etv2, Brg1 and H3K27ac post Etv2 induction in MEF and ES/EB. Methods: EB or MEF cells were harvested as described above. ChIP assays for ETV2 and H3K27ac were performed from 2 x 10e6 MEFs or 1 x 10e7 EB cells, respectively. Briefly, cells were crosslinked with 1% formaldehyde for 10 min at room temperature and the reaction was quenched by glycine at a final concentration of 0.125 M. For the BRG1 ChIP-seq, cells were first crosslinked in 2 mM disuccinimidyl glutarate (DSG; Life Technologies: Cat. #20593) for 30 min then in 1% formaldehyde for 10 min, quenched with glycine for 5 min. The remainder of the ChIP protocol was performed following the protocol described by Magli et al19. We used antibodies against H3K27ac (Abcam, ab4729), ETV2 (Abcam, ab181847), and BRG1 (Abcam ab110641) for the respective pulldowns. For both of the pulldowns, protein A Dynabeads were added to the ChIP reactions and incubated for 30 minutes at room temperature. Magnetic beads were washed and chromatin was eluted. After crosslinking reversal, RNase A, and proteinase K treatment, ChIP DNA was extracted with the Min-Elute PCR purification kit (Qiagen). ChIP DNA was quantified with Quant-it PicoGreen dsDNA Assay Kit (Life Technologies). Sequencing libraries were prepared using equal amount of ChIP DNA per sample as per instructions of SMARTer® ThruPLEX® DNA-Seq Kit (Takara R400675) and SMARTer® DNA Unique Dual Index Kit - 24U Set A (Takara R400665). Sequencing was performed at the University of Minnesota Genome Center with the The NextSeq 550 high-throughput benchtop sequencer.

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ATAC-seq analysis of Etv2 induced MEF reprogramming and EB differentiation.

Project description:ATAC-seq analysis of Etv2 induced ES/EB, MEFs,Brg1 KO EBs and Brg1 KD MEFs. Methods: EBs were collected at different time points and disaggregated in 0.25% trypsin at 37C for 3 min incubation with gentle agitation followed by inactivation with culture medium containing 10% FBS. Cells were collected by centrifugation at 500g for 5 minutes, washed once with ice-cold PBS. iHA-Etv2 MEFs were harvested from the culture dishes at different time points by treating the cells with 0.25% trypsin at 37C for 4 min incubation followed by neutralizing and collecting the cells with culture medium containing 10% FBS. ATAC reaction was performed with 50,000 cells as previously described18 using the Tn5 transposase (Illumina) and libraries were created at the University of Minnesota Genome Center. Libraries were then sequenced on a NextSeq Illumina platform (2x50 bp) aiming for 25 million reads per sample.

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Etv2-induced MEF reprogramming

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Gene expression analysis of inducible ES cells overexpressing Etv2 (induced for 12 hours at day 3 of differentiation)

Project description:During embryogenesis, the endothelial and the hematopoietic lineages first appear during gastrulation in the blood island of the yolk sac. We have previously reported that an Ets variant gene 2 (Etv2/ER71) mutant embryo lacks hematopoietic and endothelial lineages, however, the precise roles of Etv2 in yolk sac development remains unclear. We carried out a transcriptome analysis using an ES cell line that can inducibly express Etv2. Cells were induced for 12 hours at day 3 of differentiation. An ES cell line (dervied from mouse of background strain 129P2/Ola) with a doxycycline inducible expression casette of Etv2 (ER71 A2Lox.cre) was differentiated in hanging drops. At 3 days of differentiation, cells were treated with doxycycline for 12 hours, and subsequently Flk1(+) cells were sorted. Experiment was done in triplicate and untreated cells were used for control.

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RNA-seq analysis of EpiSCs undergoing differentiation induced by EB formation

Project description:To examine global gene expression changes during differentiation of epiblast stem cells (EpiSCs) induced by embryoid body (EB) formation, we performed RNA-seq analysis.

2018-06-01 | E-MTAB-5343 | biostudies-arrayexpress

Gene expression analysis of inducible ES cells overexpressing Etv2 (induced for 12 hours at day 3 of differentiation)

2012-07-04 | GSE37658 | GEO