title: ChIP-seq (H3K4me3) of mouse embryonic stem cells (+Dox, rep 2)
design: Chromatin immunoprecipitation and sequencing (ChIP-Seq) and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) experiments were carried out as in Daniel B et al. Methods Mol Biol. 2014, with minor modifications. Briefly, nuclei were cross-linked in 4% methanol-free ultrapure formaldehyde (Thermo Fisher Scientific, Waltham, Massachusetts, USA) for 10 minutes at room temperature. Glycine was added for 5 min at a final concentration of 125 mM. After fixation, chromatin was sonicated with a Diagenode Bioruptor to generate 2001000 bp fragments. Chromatin was immunoprecipitated with rabbit polyclonal anti-H3K4me3 (Abcam, Cambridge, UK; 1 mg/ml) antibody using pre-blocked magnetic beads (Dynabeads Protein A, Thermo Fischer). Eluted DNA was purified (MinElute PCR Purification Kit; Qiagen Inc., Valencia, California, USA), then quantified with a Qubit fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA). ChIP-seq libraries were prepared from two biological replicates by Illumina according to manufacturers instructions.