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SRX2402717: GSM2418860: WT_CTCF_ChIPSeq; Mus musculus; ChIP-Seq
2 ILLUMINA (Illumina HiSeq 4000) runs: 89.6M spots, 4.6G bases, 1.5Gb downloads

Submitted by: NCBI (GEO)
Study: CTCF and cohesin regulate chromatin loop stability with distinct dynamics
show Abstracthide Abstract
The study investigates the nuclear organization and the binding dynamics of CTCF and cohesin by super-resolution microscopy and ChIP-seq. We determined the genome-wide binding profile of CTCF and Rad21 cohesin subunit in wild type mouse ES cells and compared them to a double FLAG-Halo-mCTCF/mRad21-SNAPf-V5 knock-in mES line, generated by CRISPR/Cas9-mediated genome editing to perform imaging experiments. Overall design: ChIP-Seq of CTCF and Rad21 cohesin subunit in wild type mouse ES cells (WT) and in mouse ES cells homozygous for FLAG-Halo-CTCF and Rad21-SNAPf-V5 (C59 clone). ChIP-Seq libraries were prepared independently from two ChIP biological replicates, using normal serum IgGs as a control, and including total inputs as reference datasets.
Sample: WT_CTCF_ChIPSeq
SAMN06117046 • SRS1842195 • All experiments • All runs
Organism: Mus musculus
Instrument: Illumina HiSeq 4000
Strategy: ChIP-Seq
Selection: ChIP
Layout: SINGLE
Construction protocol: Cells were washed twice with ice-cold PBS, scraped, centrifuged for 10 min at 4000 rpm, resuspended in cell lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, and 0.5% NP-40, 1 ml/15 cm plate) and incubated for 10 min on ice. During the incubation, the lysates were repeatedly pipetted up and down every 5 minutes. Lysates were then centrifuged for 10 min at 4000 rpm. Nuclear pellets were resuspended in 6 volumes of sonication buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 0.1% SDS), incubated on ice for 10 min, and sonicated to obtain DNA fragments below 2000 bp in length (Covaris S220 sonicator, 20% Duty factor, 200 cycles/burst, 100 peak incident power, 50 cycles of 30" on and 30" off). Sonicated lysates were cleared by centrifugation and 400-1600 μg of chromatin was diluted in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl) to a final concentration of 0.8 μg/μl, precleared with Protein A sepharose (GE Healthcare) for 2 hours at 4°C and immunoprecipitated overnight with 8-16 μg of normal rabbit IgGs, anti-Rad21 or anti-CTCF antibodies. About 15% of the precleared chromatin was saved as input. Immunoprecipitated DNA was purified with the Qiagen QIAquick PCR Purification Kit, eluted in 60 μl of water and analyzed by qPCR together with 2% of the input chromatin prior to ChIP-Seq library preparation. ChIP-seq libraries were prepared independently from two ChIP biological replicates using the Illumina TruSeq™ DNA sample preparation kit according to manufacturer instructions with few modifications. We used 100 ng of ChIP input DNA (as measured by Fragment analyzer™) and 50 μl of immunoprecipitated DNA as a starting material; Illumina adapters were diluted 1:50, and library samples were enriched through 18 cycles of PCR amplification. We assessed library quality and fragment size by qPCR and Fragment analyzer™, and when necessary we performed an additional size selection step on agarose gel after PCR amplification to enrich for fragments between 150 and 500 bp. We sequenced 4 to 8 multiplexed libraries per lane on the Illumina HiSeq4000 sequencing platform (single end-reads, 50 bp long) at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley, supported by NIH S10 OD018174 Instrumentation Grant.
Experiment attributes:
GEO Accession: GSM2418860
Runs: 2 runs, 89.6M spots, 4.6G bases, 1.5Gb
Run# of Spots# of BasesSizePublished


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