PMC:2626671 / 31057-33611 JSONTXT

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After washes, cell pellets were resuspended at 40 × 106 cells/ml in ChIP lysis buffer (150 mM NaCl, 25 mM Tris [pH 7.5], 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate plus protease and phosphatase inhibitors), and chromatin was sheared with a sonicator to yield 0.5–1-kb DNA fragments. After preclearing the sheared chromatin with protein A–sepharose beads and removing 5% as input chromatin, immunoprecipitation was performed by adding optimized antibody amounts (per 20 × 106 cell equivalents: 2.5 μg anti-Eomes, 1:100 dilution anti-Runx3), followed by overnight incubation at 4°C; protein A–sepharose beads were added for the last 3 h of the incubation period. Beads were washed 2× with RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate), 1× with high salt buffer (50 mM Tris [pH 8], 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and 1× with TE buffer. After the last wash, DNA was eluted by resuspending the beads in elution buffer (1% SDS, 100 mM NaHCO3). Both input and ChIP chromatin were then treated with RNase A (5 μg total) for 1 h at 37°C, followed by the addition of proteinase K (100 μg total) and overnight incubation at 65°C to reverse cross-linking. DNA was then purified with QIAquick columns (Gel Extraction Kit; QIAGEN) according to the manufacturer's instructions and resuspended in a 50-μl volume. For real-time PCR detection of immunoprecipitated targets using the SYBR Green PCR Kit, a standard curve was obtained with serial dilutions of input DNA for each sample, and 1 μl ChIP DNA was used per PCR reaction (performed in duplicates). Melt curves and agarose gels were analyzed to ensure amplification of specific target sequences. Refer to Table S1 (available at http://www.jem.org/cgi/content/full/jem.20081242/DC1) for a list of primer sets. The data are presented as the number of immunoprecipitated target sequences relative to input chromatin, assuming two copies of target sequence per cell equivalent used for the 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