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Molecular profiling of Ctcf hemizygous mouse embryonic fibroblasts: RNA-seq

ABSTRACT: CTCF is a highly conserved and ubiquitously expressed protein involved in several fundamental processes such as fine-tuning gene expression, imprinting, X-chromosome inactivation and 3D chromatin organisation. To understand the impact of differences in the concentration of CTCF abundance on these processes, we exploit a CTCF hemizygous mouse model with a stable reduction in the concentration of this protein. We derived twelve independent primary lines of mouse embryonic fibroblasts (MEFs) from six wildtype and six CTCF-hemizygous mouse E13.5 embryos. Total RNA from each MEF line was purified using QIAzol Lysis Reagent (Qiagen); DNase treatment and removal was performed using the TURBO DNA-freeTM Kit (Ambion, Life Technologies). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina) and sequenced in an Illumna HiSeq4000 to produce 150bp paired-end reads. On the same MEF lines we have performed ChIPseq for CTCF, H3K4me3 and H3K27ac and HiC.

INSTRUMENT(S): Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Sarah Aitken

PROVIDER: E-MTAB-6259 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Molecular profiling of Ctcf hemizygous mouse embryonic fibroblasts: HiC

Project description:CTCF is a highly conserved and ubiquitously expressed protein involved in several fundamental processes such as fine-tuning gene expression, imprinting, X-chromosome inactivation and 3D chromatin organisation. To understand the impact of differences in the concentration of CTCF abundance on these processes, we exploit a CTCF hemizygous mouse model with a stable reduction in the concentration of this protein. We derived independent primary lines of mouse embryonic fibroblasts (MEFs) from wildtype and CTCF-hemizygous mouse E13.5 embryos. For three biological replicates, cells were fixed in DMEM containing 2% fresh formaldehyde and incubated at room temperature for 10 min, quenched with 1M glycine for 5 min, and washed twice with ice cold PBS, before being flash-frozen at -80°C. Cross-linked cells were lysed, followed by chromatin HindIII digestion, biotinylataion, ligation, proteinase K treatment, DNA purification, sonication, end repair, biotin pull-down, adapter ligation, and PCR amplification. Pooled indexed libraries were sequenced on an Illumina HiSeq4000 to produce paired-end 150bp reads. On the same MEF lines we have performed RNAseq and ChIPseq for CTCF, H3K4me3 and H3K27ac.

2018-07-29 | E-MTAB-6262 | biostudies-arrayexpress

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Project description:CTCF is a highly conserved and ubiquitously expressed protein involved in several fundamental processes such as fine-tuning gene expression, imprinting, X-chromosome inactivation and 3D chromatin organisation. To understand the impact of differences in the concentration of CTCF abundance on these processes, we exploit a CTCF hemizygous mouse model with a stable reduction in the concentration of this protein. We derived twelve independent primary lines of mouse embryonic fibroblasts (MEFs) from six wildtype and six CTCF-hemizygous mouse E13.5 embryos. MEFs were fixed in DMEM containing 1% fresh formaldehyde and incubated at room temperature for 10 min, quenched with 250mM glycine for 10 min, and washed twice with ice cold PBS, before being flash-frozen at -80°C. Cross-linked cells were lysed and sonicated on a Bioruptor Plus (Diagenode) sonicator to fragment chromatin to an average length of 300bp. 10 ug of the following antibodies were used for immunoprecipitation: CTCF (rabbit polyclonal, Merk Millipore 07-729, lot 2517762); H3K4me3 (mouse monoclonal IgG clone CMA304, Merck Millipore 05-1339, lot 2603814); H3K27ac (rabbit polyclonal IgG, Abcam 4729, lot GR244014-1). Immunoprecipitated DNA or 50 ng of input DNA was used for library preparation using the ThruPLEX DNA-Seq library preparation protocol (Rubicon Genomics, UK). Library fragment size was determined using a 2100 Bioanalyzer (Agilent). Libraries were quantified by qPCR (Kapa Biosystems). Pooled libraries were sequenced on a HiSeq4000 (Illumina) according to manufacturer’s instructions using single-end 50 bp reads. On the same MEF lines we have performed RNAseq and HiC (see related accession numbers).

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Molecular profiling of Ctcf hemizygous mouse embryonic fibroblasts: HiC

Project description:Molecular profiling of Ctcf hemizygous mouse embryonic fibroblasts: HiC

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