One × 108 cells were fixed for 10 minutes in 0.37% formaldehyde while 100 mg of tissue was roughly chopped in and fixed for 15 minutes in 1% formaldehyde. The fixation reaction was quenched with 0.125 M glycine. Tissue was then homogenized in fresh PBS. Nuclei were isolated by incubation on ice for 10 minutes in cell lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% NP-40, 10 mM sodium butyrate, and proteinase inhibitors) and lysed in nuclei lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS, 10 mM sodium butyrate, and proteinase inhibitors) for 10 minutes on ice. The lysate was sonicated to shear the chromatin, cell debris removed by centrifugation, and immunoprecipitation (IP) dilution buffer (20 mM Tris-HCl, pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.01% SDS, 10 mM sodium butyrate, and proteinase inhibitors) added to bring the ratio of nuclei lysis buffer/dilution buffer to 1:4. Chromatin was precleared for 3 hours at 4°C with 200 μl of protein-G-Sepharose blocked with 5% BSA. Whole-cell extract was divided into aliquots: 270 μl kept as input chromatin and 1.35-ml aliquots incubated with antibodies against proteins of interest. Immune complexes were collected by rotation at 4°C for 3 hours with 100 μl of blocked protein-G-Sepharose. Beads were washed twice with wash buffer 1 (20 mM Tris-HCl pH 8.1, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, and 0.1% SDS), once with wash buffer 2 (10 mm Tris-HCl pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, and 1% deoxycholic acid), and twice with TE (10 mM Tris/HCl, 1 mM EDTA) pH 8.0. Immune complexes were eluted with elution buffer (100 mM NaHCO3, 1% SDS). Cross-links were reversed by incubation at 65°C for 6 hours in the presence of 5 μg of RNase A and 0.3 M NaCl. Ninety micrograms of proteinase K was added, and samples were incubated overnight at 45°C. DNA was purified using phenol/chloroform extraction and ethanol precipitation. Input pellets were resuspended in 200 μl of water, other samples in 100 μl of water. Two-microliter aliquots were analyzed in quantitative PCR using primers designed adjacent to the RE1 region. The amount of DNA pulled down with antibodies to the proteins of interest was compared with that pulled down with the same amount of a nonspecific antibody (rabbit or goat polyclonal HA antibody; Santa Cruz Biotechnology, Inc.) or, in the case of antisera, to the same volume of normal rabbit serum (Santa Cruz Biotechnology, Inc.). The following antibodies were used: goat polyclonal P18 to REST, rabbit polyclonal to Sin3a, rabbit polyclonal to Sin3b, rabbit polyclonal to HDAC1, rabbit polyclonal to HDAC2, and rabbit polyclonal to c-myc (all Santa Cruz Biotechnology, Inc.), rabbit antiserum to CoREST, rabbit antiserum to H4Ac, rabbit polyclonal to H3K9Ac, rabbit antiserum to H3K4diMe, and rabbit antiserum to H3K4triMe (all Upstate Biotechnology, Lake Placid, NY, http://www.upstatebiotech.com), and rabbit polyclonal to H3K9diMe and rabbit polyclonal to H3 (all Abcam).