Typhoon 9410 variable imager

Recombinant yeast histones were purified as previously described (Tsukiyama Lab online protocol http://research.fhcrc.org/tsukiyama/en/protocols.html) (Luger et al. 1999 (link)) and dialyzed by gradient salt dialysis onto the Widom 601 positioning sequence (Lowary and Widom 1998 (link)) with 80 bp of linker DNA and, where indicated, a URS1 site (TGGCGGCT) located +8 to +15 with respect to the nucleosome edge and a 5′ FAM label. Purified end-positioned mononucleosomes (50 nM) were incubated with 10 nM Chd1 protein constructs in nucleosome sliding buffer (10 mM HEPES at pH 7.8, 10 mM MgCl₂, 0.1 mM EDTA, 10% glycerol, 5 mM ATP, 0.2 mg/mL BSA, and 100 mM KCl). Nucleosome sliding reactions were quenched by adding 1 µg of unlabeled competitor plasmid and placed on ice. To resolve nucleosome positions, native gels (6% acrylamide, 60:1 acrylamide:bisacrylamide ratio in 1× TBE) were run for 2 h at 150 volts at 4°C in prechilled 0.25× TBE running buffer and visualized using a Typhoon 9410 variable imager (GE Healthcare).

Streptomycin aptamer and Strep-RS7 RNAs were generated by in vitro transcription with T7 RNA polymerase and subsequent polyacrylamide (PAA) gel purification and ethanol precipitation. RNA 5΄-ends were dephosphorylated using antarctic phosphatase (NEB) and labeled with [γ-³²P]-ATP (Hartmann Analytic) using T4 polynucleotide kinase (NEB) according to manufacturer's protocols. Structural analysis of 10 000 cpm 5΄-end labeled RNA per reaction was performed via in-line probing (38 (link)) in the presence of 50 mM Tris-HCl (pH 8.5), 12 mM MgCl₂, 0.1 mg/ml tRNA from E. coli MRE 600 (Roche Diagnostics) and 0–10 mM streptomycin. All purification steps were carried out on 12.5% or 15% denaturing PAA gels. Imaging of ³²P-labeled RNA was performed with a Typhoon 9410 Variable Imager (GE Healthcare).