The assays were performed as previously reported (18, 21). Briefly, reconstituted KAT6 complexes were incubated with 0.2 to 1 μg of nucleosomes and 0.1 mM acetyl-CoA (Sigma-Aldrich, A2056), propionyl-CoA (Sigma-Aldrich, P5397), crotonyl-CoA (Sigma-Aldrich, 28007), or butyryl-CoA (Sigma-Aldrich, B1508) at 37°C for 1 hour. HeLa oligonucleosomes were purified as previously described (21), where recombinant mononucleosomes were purchased from EpiCypher (16-0009). Reactions were terminated by addition of a 3× reducing SDS sample buffer for separation by SDS–polyacrylamide gel electrophoresis (PAGE) (15%) and transferred to 0.22-μm nitrocellulose membrane (Pall Laboratory, 66485) and subsequent immunoblotting analysis to detect histone H3 or its acylation with site-specific antibodies as described below. The KAT6 complexes in the acylation reactions were verified by immunoblotting with anti-FLAG (Sigma-Aldrich, F3165) and anti-HA (BioLegend, 901515) antibodies.

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