Draiii hf

DraIII-HF (New England Biolabs (NEB), R3510S) was used to linearize the circular DNA plasmid (sequence in Supplementary Table 1) following the manufacturer’s recommendations. In a 50 µL reaction, 2000 ng of circular DNA plasmid were mixed with 2 µL of DraIII-HF (40 units), 5 µL of 10X rCutSmart Buffer, and nuclease-free water to achieve final reaction volume. The reaction components were mixed by pipetting and spin down for a couple of seconds. The reaction was incubated at 37 °C for 1 h. Enzymes were kept on ice throughout the whole preparation procedure. After DraIII-HF treatment, DNA was purified using Monarch PCR & DNA Cleanup Kit (5 μg) (NEB, T1030S).
ScaI-HF (20,000 units/mL, NEB, Catalog number R3122S) was used to linearize circular DNA plasmid for Supplementary Fig. 24 (plasmid sequence in Supplementary Table 5). A reaction of 2000 ng of DNA was prepared according to the manufacturer’s recommendations. In a 50 µL reaction, 2000 ng of circular DNA plasmid were mixed with 2 µL of ScaI-HF (40 units), 5 µL of 10X rCutSmart Buffer, and nuclease-free water to reach 50 µL. The reaction was incubated at 37 °C for 1 h. DNA was also purified after digestion with ScaI-HF.

DNA sequences used for Cga and Lhb nucleosome reconstitution were amplified by PCR from mouse genomic DNA. Sequences for 601 nucleosomes33 (link) were amplified from a plasmid that was a generous gift from Daniela Rhodes (MRC, Cambridge, UK). Primers used for the amplification reactions are listed in Supplementary Table 4. The constructs were digested using DraIII-HF (New England Biolabs) overnight according to the manufacturer's instructions. A 10 bp hairpin (Sigma) was ligated to the construct using T4 DNA ligase (New England Biolabs), in a reaction with 1:10 molar excess of the hairpin, at 16 °C. The construct was subsequently digested overnight with BglI (New England Biolabs).

To generate unwinding/translocation tracks of different lengths^{18 (link)}, 600 and 4000 bp tracks were obtained using standard PCR reactions (Supplementary Table 11, IDT) and nicked using Nt.BbvCI for the Biotin-terminated track and Nb.BbvCI for the Digoxigenin-terminated one (enzymes from New England Biolabs), resulting in complementary 29-nucleotides flanked with three nucleotides (5′-TGC-3′). For the symmetric geometry, the 600 biotin and digoxigenin tracks were mixed at equal molar ratios for DNA annealing, creating a ∼1200 bp fragment. For the asymmetric geometries, 4000 bp tracks were annealed to complementary purchased oligonucleotides with the opposite modification (Supplementary Table 11, HPLC purified, IDT). This resulted in asymmetric tracks with 4000 bps and ∼35 nt single-stranded DNA on opposite sides. A ∼250 dsDNA stem containing the “601” sequence^{48 (link)} was amplified from a plasmid (a generous gift from Daniela Rhodes, MRC, Cambridge, UK) using primers listed in Supplementary Table 11, digested using DraIII-HF (New England Biolabs) overnight according to the manufacturer’s instructions, and ligated to the tracks.