Ten μg biotinylated intasome was allowed to bind 10 μg recombinant or native nucleosomes in 700 μlof pull-down buffer (140-340 mM NaCl, 10 % glycerol, 1 mM DTT 0.1 % Nonidet P-40 and 50 mM BisTrispropane-HCl, pH 7.45), in the presence of 20 μl of streptavidin agarose (Life Technologies). After incubation for 2 h of end-over-rocking at 4 °C, the resin was washed in 5 changes of 700 μl pull-down buffer. Bound nucleoprotein complexes, dissociated by incubating the resin in 30 μl of 1.3× Laemmli SDS sample buffer at 37 °C for 5 min, were analyzed by electrophoresis in 18% SDS PAGE gels (Life Technologies). Proteins and DNA were detected by staining the gels with Coomassie instant blue and GelRed (Biotium), respectively.
To generate a library of nucleosomal DNA fragments enriched in tighter intasome binders, a biotin pull-down experiment setup with a 10-fold excess of nucleosomes in the presence of 290 mM NaCl. The DNA component of recovered nucleosomal fraction was treated with S1 nuclease and calf intestinal phosphatase (New England Biolabs). Blunt-ended and dephosphorylated DNA was incubated with Taq Polymerase to add deoxyadenosine to 3′ ends for cloning into pCR4-TOPO (Life Technologies). Individual clones were sequenced using M13 forward primer.
To generate a library of nucleosomal DNA fragments enriched in tighter intasome binders, a biotin pull-down experiment setup with a 10-fold excess of nucleosomes in the presence of 290 mM NaCl. The DNA component of recovered nucleosomal fraction was treated with S1 nuclease and calf intestinal phosphatase (New England Biolabs). Blunt-ended and dephosphorylated DNA was incubated with Taq Polymerase to add deoxyadenosine to 3′ ends for cloning into pCR4-TOPO (Life Technologies). Individual clones were sequenced using M13 forward primer.