Native chromatin immunoprecipitation (ChIP) from mouse sperm nucleosomes
Sperm nucleosomes prepared as described in this study was buffer-exchanged into IP buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 0.2 mM EDTA, 0.1% NP-40, 1 mM DTT) by PD10 (GE health care). Antibodies were bounded to Dynabeads Protein G (Life Technologies), then mixed with sperm nucleosomes. Immunoprecipitates were washed with low salt buffer (20 mM Tris-HCl pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20 mM Tris-HCl pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl buffer (20 mM Hepes-OH pH 7.4, 500 mM LiCl, 1 mM EDTA, 0.5% deoxycholate, 1% NP-40), TE 50 buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA) sequentially, then eluted with elution buffer (1% SDS in TE 50 buffer) at 65°C for 20 min. The eluates were incubated at 65°C overnight, then RNAs were degraded with RNase A (Sigma-Aldrich) at 50°C for1 h, and then proteins were degraded with Proteinase K at 50°C for 2 h. Immunoprecipitated DNAs were purified using QIAquick PCR Purification kit (Qiagen).