In vitro histone acylation assay

For the analysis of histone acylation (e.g. crotonylation), we incubated histone H3 with histone acetyltransferase (HAT) p300 and crotonylation³⁰. In vitro enzymatic assays were carried out by incubating 0.5 μg of p300 with 10 μg of recombinant histone H3 in the presence of 0.5 mM crotonyl-CoA (Sigma-Aldrich) in 1X HAT buffer (25 mM pH 8 Tris-HCl, 25 mM KCl, 1mM DTT, 0.1mM AEBSF and 5 mM sodium butyrate) for 60 min at 30°C; final volume was 50 μl. Histones were derivatized with propionic anhydride, desalted and injected into an online nanoLC-MS/MS as described above.