Ruler elements in chromatin remodelers set nucleosome array spacing and phasing

Elisa Oberbeckmann; Vanessa Niebauer; Shinya Watanabe; Lucas Farnung; Manuela Moldt; Andrea Schmid; Patrick Cramer; Craig L. Peterson; Sebastian Eustermann; Karl-Peter Hopfner; Philipp Korber

Embryonic D. melanogaster histone purification. The preparation of embryonic D. melanogaster histones octamers was carried out as described in Krietenstein et al. 2012 and Simon and Felsenfeld, 1979 . Briefly, 50 g of 0-12 hours old D. melanogaster embryos were dechorionated in 3 % sodium hypochlorite, washed with dH20 and resuspended in 40 mL lysis-buffer (15 mM K·HEPES pH 7.5, 10 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 0.2 mM PMSF, 10 % glycerol). Embryos were homogenized (Yamamoto homogenizer), filtered through cloth and centrifuged at 6,500 g for 15 min. Nuclei (brownish light pellet) were washed 3 times with 50 mL sucrose-buffer (15 mM K·HEPES pH 7.5, 10 mM KCl, 5 mM MgCl2, 0.05 mM EDTA, 0.25 mM EGTA, 1 mM DTT, 0.2 mM PMSF, 1.2 % sucrose) and resuspended in 30 mL sucrose-buffer containing 3 mM CaCl2. To obtain mononucleosomes, nuclei were incubated for 10 min at 26 °C with 6250 Units MNase (Sigma-Aldrich). Reaction was stopped with 10 mM EDTA, nuclei were pelleted and resuspended in 6 mL TE (10 mM Tris·HCl pH 7.6, 1 mM EDTA) containing 1 mM DTT and 0.2 mM PMSF followed by 30 to 45 min of rotation at 4 °C. Nuclei were centrifuged for 30 min at 15,300 g at 4 °C. Solubilized mononucleosomes are found in the supernatant, which was applied to a pre-equilibrated hydroxyapatite column. After washing the hydroxyapatite column with 0.63 M KCl, histone octamers were eluted with 2 M KCl, concentrated and stored in 50 % glycerol and 1x Complete (Roche) protease inhibitors without EDTA at -20 °C.