For standard chromatin immunoprecipitation (ChIP) assays, human ES cells and normal WI-38 fibroblasts were cross-linked at room temperature in ESC medium or incomplete WI-38 medium with formaldehyde for 10 min and 6 min, respectively. To increase the likelihood of detecting occupancy of non-DNA binding proteins, we performed dual cross-linking using two long-arm protein-protein cross-linking agents: ethylene glycol-bis(succinimidyl succinate) (EGS) and dimethyl 3,3′-dithiobispropionimidate · 2HCl (DTBP) (Thermo Scientific, Rockford, IL). Human ES cells were separately incubated with EGS or DTBP in hESC medium for 20 min at room temperature before the addition of formaldehyde. Cross-linking was stopped by the addition of glycine for 5 min at room temperature. Cells were harvested in ice-cold PBS containing 1× complete protease inhibitor (Roche Applied Science, Indianapolis, IN), 1× phosphatase inhibitor cocktail I (Calbiochem, Gibbstown, NJ), 1× phosphatase inhibitor cocktail II (Calbiochem), and 20 mM sodium butyrate (Sigma). Cells were rapidly frozen in liquid nitrogen and stored at −80°C until use. Cell pellets were resuspended in buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 0.25% Triton X-100, 1× protease inhibitors), and after centrifugation the pellet was resuspended in buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease inhibitors). The pelleted nuclei were then resuspended in buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, 1× protease inhibitors) and sonicated with a Misonix ultrasonic liquid processor S-4000 (QSonica, LLC, Newtown, CT). Chromatin was recovered from the supernatant, and the efficiency of sonication was analyzed by electrophoresis on an agarose gel to monitor the size of sheared DNA (between 250 and 650 bp). Chromatin was aliquoted, rapidly frozen in liquid nitrogen, and stored at −80°C until use. ChIP assays were initiated by diluting chromatin either in buffer FA (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1× protease inhibitors) for H3K4me3 (Abcam, Cambridge, MA) antibody or in sonication buffer 3 for the remaining antibodies. The following antibodies were used per 100 μg chromatin: HINFP (5 μl of 802K rabbit polyclonal antiserum) (40), RNA polymerase II (pol II) (2.5 μl of 8WG16 mouse monoclonal IgG2a, ascites fluid; catalog no. MMS-126R; Covance [Princeton, NJ]), p220NPAT (5 μl of rabbit polyclonal serum kindly provided by J. Wade Harper, Harvard Medical School , or 5 μg of mouse monoclonal IgG2b; catalog no. 611344; BD Biosciences [San Jose, CA]), LSM10 (2 μg of mouse monoclonal IgG2a; catalog no. BMR00504; Bio Matrix Research Inc. [Nagareyama City, Chiba, Japan]), LSM11 (10 μl of rabbit affinity-purified antibody ), FLASH (12 μl of rabbit affinity-purified antibody ), SLBP (10 μl of rabbit polyclonal antibody ), CPSF73 (1 μg of rabbit affinity-purified antibody; catalog no. A301-091A; Bethyl Laboratories, Inc. [Montgomery, TX]), histone H3 (5 μg of rabbit polyclonal IgG; catalog no. ab1791; Abcam), H3K4me3 (5 μl of rabbit polyclonal serum; catalog no. 39159; Active Motif [Carlsbad, CA], or 20 μg of mouse monoclonal IgG2b; catalog no. ab1012; Abcam), H3K9ac (5 μg of rabbit polyclonal IgG; catalog no. ab4441; Abcam), H3K27me3 (5 μg of rabbit polyclonal IgG; catalog no. 07-449; Millipore [Billerica, MA]), H4K12ac (5 μl of rabbit polyclonal serum; catalog no. 07-595; Millipore), and H4K16ac (5 μg of rabbit polyclonal serum; catalog no. 07-329; Millipore). After incubation with the antibodies overnight at 4°C, chromatin-antibody complexes were recovered by incubating with preblocked protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at 4°C on a rotating wheel. Beads were washed, DNA was eluted, and cross-links were reversed. DNA was recovered by ethanol precipitation and analyzed by quantitative PCR (qPCR). ChIP results were expressed as percent input. Alternatively, DNA concentration was quantified by fluorometry (Qubit, Invitrogen), and all samples were adjusted to the same concentration. DNA (0.5 ng) was analyzed by qPCR and expressed as fold enrichment (2−[ChIP CT − input CT]). Full paper
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