Proteinase inhibitors complete

Mouse hippocampus was harvested immediately after euthanasia. Chromatin immunoprecipitation was then performed as described in Broad ChIP protocol (http://www.roadmapepigenomics.org/protocols/type/experimental/). Briefly, tissues were minced and crosslinked in 1% formaldehyde (Thermo Scientific) for 15 min at room temperature and quenched with glycine for 5 min (Sigma). The samples were homogenized in cell lysis buffer containing proteinase inhibitors (complete, Roche) and chromatin was then fragmented to a size range of ~200–500 bp using a Branson 250 digital sonifier. Solubilized chromatin was then diluted and incubated with ~1 μg antibody at 4°C overnight. Immune complexes were captured with Protein A-sepharose beads, washed and eluted. Enriched chromatin was then subjected to crosslink reversal and proteinase K digestion at 65°C, phenol-chloroform extraction and ethanol precipitation. Isolated ChIP DNA was resuspended and quantified using the Qubit assay (Invitrogen). H3K4me1 (Abcam, ab8895), H3K4me3 (Millipore, #07-473), H3K9me3 (Abcam, ab8898), H3K27me3 (Millipore, #07-449), H3K27ac (Abcam, ab4729), H3K36me3 (Abcam, ab9050) and H4K20me1 (Abcam, ab9051) were used to immunoprecipitate endogenous proteins.