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

For low, medium and high assembly degrees, 10 µg of plasmid library DNA (S. cerevisiae, S. pombe or E. coli) was mixed with ~2, 4 or 8 µg of Drosophila embryo histone octamers, respectively, in 100 µl assembly buffer (10 mM Tris·HCl, pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05 % IGEPAL CA630, 0.2 µg BSA). Samples were transferred to Slide-A-lyzer mini dialysis devices, which were placed in a 3 L beaker containing 300 mL of high salt buffer (10 mM Tris·HCl pH 7.6, 2 M NaCl, 1 mM EDTA, 0.05 % IGEPAL CA630, 14.3 mM β-mercaptoethanol), and dialyzed against a total of 3 L low salt buffer (10 mM Tris·HCl pH 7.6, 50 mM NaCl, 1 mM EDTA, 0.05 % IGEPAL CA630, 1.4 mM β-mercaptoethanol) added continuously via a peristaltic pump over a time course of 16 h while stirring. βmercaptoethanol was added freshly to all buffers. After complete transfer of low salt buffer, samples were dialyzed against 1 L low salt buffer for 1 h at room temperature. DNA concentration of the SGD chromatin preparations was estimated with a DS-11+ spektrophotometer (Denovix) and could be stored at 4 °C for several weeks. To estimate the extent of the assembly degree, an aliquot of the sample was subjected to MNase digestion (as described below) for MNase-ladder read out.