Plasmid pull-down

Plasmid pull-downs were performed as described previously (Budzowska et al., 2015). Briefly, 6 μL Streptavidin-coupled magnetic beads (Dynabead M-280, Invitrogen) per pull-down reaction, were equilibrated with wash buffer 1 (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1mM EDTA pH 8, 0.02% tween-20) and then incubated with 12 pmol of biotinylated LacI at RT for 40 min. The beads were washed four times with pull-down buffer 1 (10 mM HEPES pH 7.7, 50 mM KCl, 2.5 mM MgCl₂, 250 mM sucrose, 0.25 mg/mL BSA, 0.02% Tween-20) and resuspended in 40 μL and stored on ice. At the indicated time points, 8 μL of reaction was added to the beads and rotated for 30 min at 4°C. The beads were subsequently washed twice in wash buffer 2 (10 mM HEPES pH 7.7, 50 mM KCl, 2.5 mM MgCl₂, 0.25 mg/mL BSA, 0.03% Tween 20) and resuspended in 2x Laemmli sample buffer.