In vitro histone methyltransferase assay

Unless indicated otherwise, each 10 µl reaction contained 600 nM PRC2, 600 nM mononucleosome, and 12 µM S-[methyl-¹⁴C]-adenosylmethionine (PerkinElmer NEC363050UC) in Covfefe buffer (50 mM Tris-HCl pH 8.0 at 30°C, 100 mM KCl, 2.5 mM MgCl₂, 0.1 mM ZnCl₂, 2 mM 2-mercaptoethanol, 0.1 mg/ml bovine serum albumin, 5% v/v glycerol). Reactions were incubated for 1 h at 30°C and stopped by adding 4× loading dye. Each reaction was then heated at 95°C for 5 min and loaded onto either 4–12% Bis-Tris gel (ThermoFisher NP0322BOX) or 10–20% Tris-Glycine gel (ThermoFisher XP10202BOX). Gels were first stained by Coomassie and scanned, then vacuum dried for 60 min at 80°C. Signal was acquired with a Typhoon Trio phosphorimager (GE Healthcare). Densitometry and analysis were carried out with ImageQuant software (GE Healthcare).

In testing RNA inhibition of histone methyltranferase activity, the reaction was set up as described above except 2.4 µM mononucleosome was used and RNA was titrated into the reaction from 60 µM (2-fold dilutions).