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

Purification of S. cerevisiae Reb1 was essentially carried out as described in . Briefly, using BY4741 genomic S. cerevisiae DNA the coding sequence for Reb1 was amplified by PCR and cloned into pET21b (Novagen) via InFusion cloning (Clontech) with a Streptavidin tag at the C terminus. Correct sequences were verified via Sanger sequencing (GATC Services at Eurofins Genomics). Expression plasmids were transformed into BL21 (DE3) cd + cells. Three liters of LB medium supplemented with 600 mg/L ampicillin were inoculated with 200 mL pre-culture. Cells were grown at 37 °C to an OD600 of 0.6 (WPA CO8000 cell density meter). Induction was carried out by addition of IPTG to a final concentration of 1 mM. Cells were grown overnight at 18 °C, harvested by centrifugation (3,500 rpm, Sorvall Evolution RC) and stored at -80 °C. Cells were resuspended in lysis buffer (50 mM Tris·HCl pH 7.9, 500 mM NaCl, 7 % glycerol, 1 mM DTT, 7 % sucrose and protease inhibitor (SIGMAFAST TM protease inhibitor cocktail, 1:100), sonicated (Branson Sonifier 250, 5 min at 40-50 % duty cycle and output control 4) and cleared by centrifugation (Sorvall Evolution RC, SS34 rotor, 15,000 g). The supernatant was dialyzed over night against 2 L low salt buffer (25 mM K·HEPES pH 8.0, 150 mM KCl, 7 % glycerol, 4 mM MgCl2, 1 mM DTT). Cation ion exchange chromatography (HiTrap SP HP 5 mL, elution buffer: 25 mM K·HEPES pH 8.0, 1 M KCl, 7 % glycerol, 4 mM MgCl2, 1 mM DTT) followed by size exclusion chromatography (Superdex 200 10/300, buffer: 25 mM K·HEPES pH 8.0, 200 mM KCl, 7 % glycerol, 4 mM MgCl2, 1 mM DTT) were used for purification. Peak fractions were analyzed by Coomassie SDS-PAGE. Fractions containing Reb1 were pooled, concentrated and stored at -80 °C.