Nucleosome extraction

To isolate nuclei, HEK cell pellets were resuspended in lysis buffer (20 mM HEPES, pH 7.5, 250 mM sucrose, 4 mM MgCl₂, 0.5% NP-40, 0.4 mM PMSF and 5 mM TCEP), homogenized with a dounce (Wheaton), then centrifuged at 2000 × g for 15 minutes at 4 °C. The nuclear pellet was suspended in wash buffer (20 mM HEPES, pH 7.5, 250 mM sucrose, 4 mM MgCl₂, 0.4 mM PMSF and 5 mM TCEP), centrifuged and nuclei were resuspended in wash buffer. To 0.5 ml of suspended nuclei, 20 ml of 0.65 M NaCl-sodium phosphate buffer (0.65 M NaCl, 50 mM phosphate, pH 6.8, 0.4 mM PMSF and 5 mM TCEP) was added dropwise with constant stirring, then vortexed for 1 hour at 4 °C. The nuclear lysate was passed through a column loaded with pre-wet Hydroxylapatite Fast Flow (Calbiochem) then washed with 0.65 M NaCl-sodium phosphate buffer. Nucleosomes were isolated with elution buffer (2.5 M NaCl, 50 mM phosphate, pH 6.8, 0.4 mM PMSF and 5 mM TCEP). Fractions containing nucleosomes were pooled and buffer-exchanged (HiPrep Desalting, GE Healthcare) into 50 mM sodium phosphate, pH 6.8. Pooled fractions were concentrated to 1 mg/ml by centrifugation (Amicon Ultra, Millipore) and stored at −20 °C.