D1n2e

Cells were lysed in NETN buffer (150 mM NaCl, 20 mM TRIS-HCl pH 8.0, 0,5 mM EDTA, 0.5% NP-40) on ice for 30 minutes with intermittent agitation followed by sonication (QSonica Q800R3) for a total of 10 minutes at 4°C (20-second pulse on/30-second pulse off at 100% amplitude). Samples were then centrifuged at 4°C for 10 minutes at 17,000 × g. The supernatant was moved to a fresh Eppendorf tube and a portion of the sample was removed to use as the input. 3 ug of antibody (ATRX, Cell Signaling Technology D1N2E; PML, Santa Cruz Biotechnology PG-M3; IgG, Bethyl Laboratories) was added to the remainder of each sample and incubated overnight at 4°C with gentle shaking. The following day the samples were incubated with Protein A (ATRX) or Protein G (PML) coated magnetic beads (Thermo Fisher Scientific 10001D, 10003D) for 30 minutes at room temperature. Beads were collected using magnetic separation, and subsequently washed in NETN buffer twice for 5 minutes each. Finally, beads were resuspended in 2X SDS sample buffer and boiled for 5 minutes before analysis by SDS PAGE.

The following antibodies and probes were used where indicated. ATRX (Cell Signaling, D1N2E), ATRX (Santa Cruz H-300), DAXX (Cell Signaling 25C12), FLAG-M2 (Sigma F1804), GAPDH (Santa Cruz, 0411), Histone H3.3 (Abcam, EPR17899), Histone H4 (Active Motif, 39269), PML (Santa Cruz H-238), PML (Santa Cruz PG-M3), α-Tubulin (Cell Signaling, 11H10), Purified Rabbit IgG (Bethyl Laboratories P120-101), Alexa Fluor 488 conjugated Donkey Anti-Rabbit IgG (Jackson ImmunoResearch), Cy3 conjugated Donkey anti-Rabbit (Jackson ImmunoResearch), Peroxidase conjugated Goat Anti-Mouse IgG (Jackson ImmunoResearch), Peroxidase conjugated Goat Anti-Rabbit IgG (Jackson ImmunoResearch). PNA Telomere probe (TelC-Cy3, PNA Bio Inc.) Flag-DAXX/pRK5 was a gift from Xiaolu Yang (Addgene plasmid #27974).