Histone H3.3K36me2/3 Peptide Pulldown Assay

Peptide pull-down assay was performed as described previously with slight modifications^{4 (link),65 (link)}. Briefly, biotinylated histone peptide, which contains histone H3.3 amino acids 27–46 with di- or tri-methylated Lys-36 (i.e. H3.3K36me2/3), was incubated with NeutrAvidin agarose (Thermo Fisher, 29204) in PBS buffer for 4 h. The peptide-resin was then washed three times with 1 mL of washing buffer (PBS plus 0.1% TritonX-100) to remove unbound peptide before overnight incubation at 4 °C with the whole cell lysate prepared from 293T cells stably expressing ZM (lysate in a final IP175 buffer as described above). After binding, the resin was washed extensively in IP175 buffer for five times and analyzed by western blot.