For total cell extracts, cell pellets were lysed using S300 buffer and incubated on ice for 5 min. After incubation, samples were sonicated for 5 min (30 s on +30 s off) using Bioruptor Pico (Diagenode) at 4°C. Total protein concentration of each sample was determined using Bradford reagent (Sigma-Aldrich) and then normalized across samples. Samples were then mixed 1:4 with 4X Laemmli and boiled for 5 min. For chromatin extract, cell pellets were incubated with S300 buffer for 5 min on ice and then centrifuged at 20,000g for 15 min at 4°C. The pellet obtained was washed once with buffer S300 and centrifuged at 20,000g for 15 min at 4°C. The pellet was then resuspended in 2X Laemmli, boiled for 10 min, then sonicated for 5 min (30 s on +30 s off) using Bioruptor Pico (Diagenode) at room temperature. The sample was then boiled again for 5 min. SDS–polyacrylamide gel electrophoresis and Western blotting were performed as previously described (30). Primary antibodies used were the as follows: 1:1000 anti-TRRAP antibody (EPR14952) (#ab183517, Abcam), 1:1000 anti-BRD8 antibody (#A300-219A-M, Bethyl), 1:1000 anti-DMAP1 (#PA1-886, Thermo Fisher Scientific), 1:1000 anti-SUZ12 (#sc-46264, Santa Cruz), 1:1000 anti-EZH2 (#5246, Cell Signaling Technology), 1:1000 anti-FLAG (#F3165, Sigma-Aldrich), 1:1000 anti-HA (HA-Tag #3724, Cell Signaling Technology), 1:1000 anti-H3K27me2 (#9728, Cell Signaling Technology), 1:1000 anti-H3K27me3 (#9733, Cell Signaling Technology), 1:1000 H3K27ac (#8173, Cell Signaling Technology), 1:1000 anti-CXORF67 (#HPA004003, Sigma-Aldrich), or affinity-purified anti-CATACOMB (1 μg/ml) (homemade rabbit polyclonal raised against a C-terminal region of the protein), 1:1000 anti-JARID2 (#13594, Cell Signaling Technology), 1:1000 anti-AEBP2 (#14129, Cell Signaling Technology), 1:1000 anti–β-tubulin (#sc-9104, Santa Cruz), 1:1000 H3K27me1 (#07-448, Sigma-Aldrich), and 1:1000 anti-H3 (#14269S, Cell Signaling Technology). Appropriate secondary antibodies according to primary hosts were used as previously described (30).

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