ChIP-seq analysis of histone modifications and CCCTC-binding factor (CTCF) binding in the livers of five strains of mouse and rat
ChIP-seq analysis of histone modifications and CCCTC-binding factor (CTCF) binding in the livers of five strains of mouse and rat
Livers from males were perfused in situ with PBS and then dissected, minced, cross-linked using 1% formaldehyde solution for 20 min, quenched for 10 min with 250 mM glycine, washed twice with ice-cold PBS, and tissue pellets were stored at -80°C. Livers (ten livers pooled for mouse or one liver for rat) were homogenised, washed twice with PBS, and lysed according to published protocols (Schmidt et al. 2009). Chromatin was sonicated to an average fragment length of 300 bp. Chromatin was immunoprecipitated using either 10 μl H3K4me3 antibody (mouse monoclonal, Merck Millipore 05-1339, lot 2780484); 10 μl H3K27ac antibody (rabbit polyclonal, AbCam 4729, lot GR3187598-1); 20 μl CTCF antibody (rabbit polyclonal, Merck Millipore 07-729, lot 2517762). Immunoprecipitated DNA or input DNA (max 50 ng) was used for library preparation using the ThruPLEX DNA-Seq library preparation protocol (Rubicon Genomics, UK) as described previously (Aitken et al. 2018). Library fragment size was determined using a 2100 Bioanalyzer (Agilent).