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SRX3200424: GSM2788283: iRx_noDoxJQ1_RNAseq_rep1; Mus musculus; RNA-Seq
1 ILLUMINA (Illumina HiSeq 2500) run: 30.2M spots, 4.6G bases, 1.7Gb downloads

Submitted by: NCBI (GEO)
Study: System-wide Dissection of the Transcriptional Response to RUNX1 During Hematopoietic Specification [RNA-seq]
show Abstracthide Abstract
The specification of hematopoietic cells in the developing embryo occurs in specific stages and is regulated by the successive establishment of specific transcriptional networks. However, the molecular mechanisms of how the different stages switch from one to another are still not well understood. Hematopoietic cells arise from endothelial cells within the dorsal aorta which transit into hematopoietic cells by a process called the endothelial-hematopoietic transition (EHT) which does not involve DNA replication. The transcription factor RUNX1 is essential for this process. Using the differentiation of mouse embryonic stem cells carrying an inducible version of RUNX1, we have previously shown that hematopoietic genes are primed prior to the EHT by the binding of transcription factors required to form both endothelial and hematopoietic cells (FLI-1 and SCL/TAL1). We demonstrated that after induction RUNX1 reshapes the transcription factor binding landscape by causing a relocation of these factors and pulling them towards its binding sites. In the study presented here, we employed the same system to globally dissect the transcriptional processes that underlay the EHT. We demonstrate that the RUNX1-mediated movement of FLI-1 involves the recruitment of the basal transcription components CDK9 and BRD4 to promoters. The looping factor LDB1 to binds to distal elements and after induction relocates towards RUNX1/FLI-1 to form a co-localizing complex in chromatin. This entire process is blocked by treatment with the BRD4 inhibitor JQ1. Our study constitutes a paradigm for transcriptional processes driving transitions in cellular shape and function which are widely observed in development and disease. Overall design: RNA-seq expreiments have been used to study RUNX1 transcription factor during Hematopoietic specification
Sample: iRx_noDoxJQ1_RNAseq_rep1
SAMN07674697 • SRS2527422 • All experiments • All runs
Organism: Mus musculus
Library:
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNA was extracted from cells using TRIzol (Invitrogen) according to manufacturer’s instructions. First-strand cDNA synthesis was carried out using Superscript II (Invitrogen) and Oligo dT (Invitrogen) according to manufacturer’s instructions. RNA was DNase treated using Turbo DNase (Ambion) and a further clean-up step was performed using the RNA Nucleospin kit (Macherey-Nagel) according to manufacturer’s instructions. Real-time PCR was carried out using Applied Biosystems SYBR green master mix (Thermo Fisher) with 5 μl of diluted cDNA and 0.25 μM forward and reverse primers per 10 μl reaction on an ABI 7900HT machine. Analysis was carried out on samples measured in duplicate. RNA-seq libraries were prepared using the Tru-seq Stranded Total RNA kit (Illumina), according to manufacturer’s instructions. Libraries were sequenced in a pool of 12 indexed libraries using a NextSeq® 500/550 High Output Kit v2 (150 cycles) for paired end sequencing (Illumina, FC-404-2002) at the Genomics Birmingham sequencing facility.
Experiment attributes:
GEO Accession: GSM2788283
Links:
Runs: 1 run, 30.2M spots, 4.6G bases, 1.7Gb
Run# of Spots# of BasesSizePublished
SRR605348530,181,5694.6G1.7Gb2018-07-19

ID:
4505946

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