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

Tandem affinity purification of ISW1a (TAP-Ioc3) and Fun30 (TAP-Fun30) was performed as follows: Cultures were grown in YPD media, harvested cells were washed once with water. The cells were lysed in buffer E (20 mM Na·HEPES pH 7.5, 350 mM NaCl, 10 % glycerol, 0.1 % Tween, and 0.5 mM DTT) and protease inhibitors by grinding in the presence of liquid nitrogen. Lysates were clarified at 40,000 g at 4 °C for 1 h. Cleared lysates were incubated with IgG-Sepharose (GE Healthcare) at 4 °C for 2 h and eluted by TEV protease (Invitrogen) cleavage at 4 °C overnight. The elutions were incubated with calmodulin affinity resin (Agilent Technology) in buffer E plus 2 mM CaCl2 at 4 °C for 2 h and eluted in buffer E plus 10 mM EGTA. ISW2 (FLAG-Isw2) was purified as follows: Cleared lysate was incubated with Anti-FLAG M2 affinity gel (Sigma-Aldrich) at 4 °C for 1 h and eluted with 0.1 mg/mL 3X FLAG peptide (Sigma-Aldrich). E-buffer (20 mM Na·HEPES pH 7.5, 350 mM NaCl, 10 % glycerol, 0.1 % Tween, and 0.5 mM DTT) was used during the entire purification. Purified proteins were concentrated with VIVASPIN concentrators (Sartorius) and dialyzed against E-Buffer with 1 mM DTT. Subunit compositions were confirmed by SDS-PAGE ( Figure S1A ) and mass spectrometry.