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The protocol for nuclei isolation and acid extraction is adapted from previous work [49]. In brief, nuclei are isolated with an NP-40 alternative based procedure in the presence of a cocktail of inhibitors. The isolated nuclei are acid extracted using 0.2 M H2SO4 to precipitate non-histone proteins and 0.2 g/ml trichloroacetic acid to precipitate a crude histone preparation of only chromatin bound histones. Histone H4 is further purified by reversed phase HPLC prior to LC-MS/MS analysis.
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