E-MTAB-6261
Molecular profiling of Ctcf hemizygous mouse embryonic fibroblasts: ChIP-seq
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).
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).