Dimethyl Sulfate (DMS) Footprinting

DMS footprinting experiments were performed as follows^39,52,68. 3′-6-FAM labelled oligonucleotides (^S1–^S3, Supporting Information Table ^S1) were diluted to 0.1 μM in 100 μl 30 mM Tris-HCl (pH 7.4) in the presence of H₂O, 100 mM LiCl or KCl. DNA samples were then annealed at 90 °C for 10 min and slowly cooled down to 4 °C (more than 8 h) before being treated with 10% (v/v) DMS for 1.5 min at 25 °C. The reaction was quenched by adding 100 μl stop solution (20 μl β-mercaptoethanol, 40 μl 3 M sodium acetate, 50 μg sperm DNA). The DMS-treated DNA samples were extracted with a Tris-phenol-chloroform solution (pH 8.0) and then precipitated using ethanol at −80 °C before being cleaved by 10% (v/v) piperidine at 90 °C for 30 min. The cleaved DNA samples were separated on a 20% sequencing gel at 1500 v for 4 h in a 4 °C cold room and imaged using a GE Healthcare Typhoon9400 gel scanner (CT, USA).