text: ChIP and real-time PCR analysis.
  20 x 106 CD8+ T cells per immunoprecipitation were fixed by adding a 1/10th volume of fixation solution (11.1% formaldehyde, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM Hepes) to 1 volume of culture media and were incubated for 10 or 30 min at RT. Fixation was stopped with 120 mM glycine on ice for 5 min. Fixed cells were washed 2x with cold PBS, 1x with cold solution I (10 mM Tris [pH 7.5], 10 mM EDTA, 0.5 mM EGTA, 1% Triton X-100), and 1x with cold solution II (10 mM Tris [pH 7.5], 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). After washes, cell pellets were resuspended at 40 x 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 x 106 cell equivalents: 2.5 mug anti-Eomes, 1:100 dilution anti-Runx3), followed by overnight incubation at 4degreesC; protein A-sepharose beads were added for the last 3 h of the incubation period. Beads were washed 2x with RIPA buffer (50 mM Tris [pH 8], 150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% deoxycholate), 1x with high salt buffer (50 mM Tris [pH 8], 500 mM NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS), and 1x 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 mug total) for 1 h at 37degreesC, followed by the addition of proteinase K (100 mug total) and overnight incubation at 65degreesC 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-mul 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 mul 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 ChIP. 
  