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

Expression and purification of INO80 complex and respective mutants. Exact strategy for recombinant expression of S. cerevisiae INO80 complex in insect cells and complex purification is described in the accompanying paper Krietenstein et al. Briefly, MultiBac technology (Trowitzsch et al., 2010) was applied to generate two baculoviruses carrying coding sequences for S. cerevisiae Ino80 (2xFlag), Rvb1, Rvb2, Arp4, Arp5-His, Arp8, Actin, Taf14, Ies1, Ies2, Ies3, Ies4, Ies5, Ies6 and Nhp10 which were subcloned into pFBDM vectors and sequence verified by Sanger Sequencing (GATC Services at Eurofins Genomics). High Five (Hi5) insect cells (BTI-TN-5B1-4 Invitrogen) were co-infected with two or three baculoviruses 1/100 (v/v) each for expression purposes. The recombinantly expressed INO80 complex and respective INO80 mutant complexes were purified from insect cells according to (Tosi, Haas et al. 2013) , which resulted in a pure and monodisperse sample. Shortly, cells were resuspended in lysis buffer (50 mM Tris·HCl pH 7.9, 500 mM NaCl, 10 % glycerol, 1 mM DTT, SIGMAFASTTM protease inhibitor cocktail), sonified (Branson Sonifier, 3x 20 s with 40 % duty cycle and output control 3-4) and cleared by centrifugation (Sorvall Evolution RC, SS34 rotor, 15,000 g). The supernatant was incubated with anti-Flag M2 Affinity Gel (Sigma-Aldrich) and centrifuged for 15 min at 1,000 g and 4 °C. Figure 4D ), one N-author/funder. All rights reserved. No reuse allowed without permission.