Chromatin immunoprecipitation (ChIP)

Immunoprecipitation of micrococcal nuclease-digested, native chromatin coupled to quantitative PCR (qPCR) was used to determine levels of histone modification at selected genomic regions, as previously described [65, 66].

Immunoprecipitation of formaldehyde cross-linked chromatin (X-ChIP) in MEFs was carried out essentially as described [67, 68]. Briefly, MEFs were cross-linked with 1% para-formaldehyde in medium for 10 min at RT. The reaction was quenched by the addition of 200 mM glycine before washing the cells twice with ice-cold PBS. Cells were resuspended in lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8, complete inhibitor cocktail(Roche)] to a concentration of 10⁵ cells in 500 μl and sonicated at high power with 30 s ON/OFF for 7 cycles (Bioruptor, Diagenode). The chromatin was diluted 1:2 with dilution buffer [1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8, 150 mM NaCl, complete inhibitor cocktail (Roche)]. Protein G Dynabeads (Thermo Fischer) were prepared resuspended in citrate–phosphate buffer (0.1 M citric acid, 0.1 M Na₂HPO₄) with 0.5% BSA. Beads were coated with antibodies for 2 h at 4 °C. The beads were washed twice, added to the chromatin solution and incubated for 4 h at 4 °C. The beads were washed twice with low-salt buffer (1% Triton X-100, 0.1% SDS, 2 mM EDTA, 150 mM NaCl, 20 mM Tris–HCl, pH 8) and twice with high-salt buffer (1% Triton X-100, 0.1% SDS, 2 mM EDTA, 100 mM NaCl, 20 mM Tris–HCl, pH 8). The bound material was eluted with Elution buffer (100 mM NaHCO₃, 1% SDS decross-linked, and the DNA extracted with QIAquick PCR purification kit (QIAGEN). Primer pairs used for analysing ChIP DNA by qPCR are listed in Additional file 5.