GSM2077299: Fetal liver Knockout of alpha globin regulatory element R... - SRA

SRX1608858: GSM2077299: Fetal liver Knockout of alpha globin regulatory element R2, biological replicate 1 [ATAC-seq]; Mus musculus; OTHER
1 ILLUMINA (NextSeq 500) run: 17.7M spots, 1.4G bases, 566.5Mb downloads

Submitted by: NCBI (GEO)

Study: Dissection of a super-enhancer in vivo (ATAC-seq)

PRJNA313964 • SRP071035 • All experiments • All runs

show Abstracthide Abstract

Many genes determining cell identity are regulated by a set of enhancer elements collectively referred to as super-enhancers. It has been suggested that super-enhancers represent a new class of cis-element, in which the assembly of enhancers confers an emergent property from the extended regulatory domain. To investigate this, we used published criteria to define one of the strongest super-enhancers in mouse erythroid cells – a 24kb region regulating a-globin expression, which comprises five enhancer-like components. Using homologous recombination, we deleted each component of this super-enhancer, singly and in informative combinations, and examined hematologic phenotype, gene expression, chromatin structure and chromosome conformation. Each component behaves independently, in an additive rather than synergistic manner. We conclude that the sub-classification of enhancers is unjustified beyond a description of their strength, which is defined by the number of lineage-specific transcription factors they bind. These findings ask afresh why enhancer-like elements cluster at key genes. Overall design: ATAC-seq on 18 Samples with different enhancer-like components

Sample: Fetal liver Knockout of alpha globin regulatory element R2, biological replicate 1 [ATAC-seq]

SAMN04526277 • SRS1318390 • All experiments • All runs

Organism: Mus musculus

Library:

Instrument: NextSeq 500

Strategy: OTHER

Source: GENOMIC

Selection: other

Layout: PAIRED

Construction protocol: A single cell suspension was made by gently dissociating the E14.5 fetal livers and passing it through a 70um filter. Ter119 selection was performed by staining the cells with phycoerythrin conjugated anti-ter119 antibodies (BD biosciences)). The cells were subsequently washed and then incubated with MACS anti-phycoerythrin beads. Cells were then separated using a magnetic column, MACS (miltenyi biotec). Nuclei were isolated by lysing the cells as previously published (Buenrostro, 2013). Assay for transposition of active chromatin sequencing (ATAC-Seq) was performed as previously published (Buenrostro, 2013). 60000-80000 cells were used per biological replicate. Cells were lysed and nuclei were isolated prior to transposition with Tn5 transposase (Nextera, Illumina) for 30 minutes at 37°C. DNA was purified using a MinElute kit (Qiagen). Libraries were amplified and barcoded using the NEBNext 2xMastermix (NEB) and the custom primers as published in Buenrostro et al., 2013. ATAC-Seq libraries profiles were visualized using D1000 tape on the Tapestation (Agilent). Due to the broad range of DNA fragment sizes found in these libraries, quantitation with the Qubit for DNA concentration was found to be highly variable and was omitted. The libraries were quantified using the universal library quantification kit (KAPA Biosystems). Samples were sequenced using: 75bp paired end reads (MiSeq platform) or 40 bp paired end reads (NextSeq platform). The strategy was to isolate open chromatin fragments specifically as they underly regulatory genomic regions. These are short stretches of DNA, with low abundance in the genome and thus require to be processed efficiently. The use of the transposase is ideal as the modified Tn5 can in one step identify the open chromatin fragments, cut them from the chromatin environment and ligate adaptors. The fragments can then be multiplexed and sequenced

Experiment attributes:

GEO Accession: GSM2077299

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 17.7M spots, 1.4G bases, 566.5Mb

Run	# of Spots	# of Bases	Size	Published
SRR3198742	17,718,811	1.4G	566.5Mb	2016-06-02

ID:: 2299664

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