P-MTAB-26367 - P-MTAB-26367
After stimulation, the cells were fixed by addition of 37% formaldehyde to a final concentration of 1%. Incubation time was 10 min at room temperature. Glycine was added to a final concentration of 125 mM for 5 min. After two washes with ice-cold phosphate-buffered saline, the cells were pelleted for 5 min at 1200 g. Pellets were resuspended in cold hypotonic lysis buffer [5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% (v/v) NP40, and protease inhibitor cocktail (Sigma)] at a ratio of 1 ml per 10^7 cells and incubated on ice for 20 min. Nuclei were pelleted by centrifugation as before and resuspended in radioimmunoprecipitation assay buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% (v/v) NP40, 1% sodium deoxycholate, 0.1% (w/v) SDS, 1 mM EDTA, and protease inhibitors] at a ratio of 1 ml per 10^7 nuclei. Soluble chromatin was prepared by sonication with a microtip using a sonifier (S-250D; Branson Ultrasonics Corporation, Danbury, CT) set to 1-s pulse, 2-s pause. Sixty pulses were applied. After centrifugation at 16,000g for 15 min in a tabletop centrifuge, the supernatant was collected. An aliquot was incubated overnight with proteinase K and RNase A at 65 degrees C and loaded on a 1% agarose gel to estimate shearing efficiency. The supernatant was precleared by addition of protein A Sepharose beads (Invitrogen), which were previously blocked in radioimmuno- precipitation assay buffer with 1 mg/ml bovine serum albumin, 0.4 mg/ml sonicated salmon sperm DNA (Stratagene), and protease in- hibitors, coupled to rabbit IgG (Sigma I5006). After rotation for 1 h at 4 degrees C, the beads were removed by centrifugation. The supernatant was used for immunpreciptiations. Four micrograms of antibody were added to 300 microliter of precleared chromatin corresponding to 3 x 10^6 nuclei and incubated overnight at 4 degrees C with mild rotation. After addition of blocked Sepharose beads, incubation time was 1 h at 4 degrees C with mild rotation. The beads were washed once with 1 ml of chilled mixed micelle buffer [20 mM Tris, pH 8.1, 150 mM NaCl, 2 mM EDTA, 0.1% (w/v) SDS, and 1% (v/v) Triton X-100], once with buffer 500 [20 mM Tris, pH 8.1, 500 mM NaCl, 2 mM EDTA, 0.1% (w/v) SDS, and 1% (v/v) Triton X-100], twice with LiCl detergent buffer (10 mM Tris, pH 8.1, 250 mM LiCl, 1% (v/v) NP40, 1% (w/v) sodium deoxycholate, and 1 mM EDTA), and twice with room-temperature Tris-EDTA. Complexes were eluted twice with 25 microliter of elution buffer [1% SDS (w/v) and 100 mM NaHCO3]. Supernatants were pooled; adjusted to 180 mM NaCl, 35 mM Tris, pH 6.5, and 9 mM EDTA; and incubated with 20 microgram of proteinase K and 1 microgram of RNase A for 65 degrees C overnight. DNA was purified using a PCR purification kit (Qiagen, Hilden, Germany).