Neb t4 ligase

45 μl of 100 μM FragA1 oligo (Supplementary Table S1), 45 μl of 100 μM FragA2 oligo (Supplementary Table S1), and 10 μl of 10× Annealing Buffer (1 M potassium acetate; 300 mM HEPES–KOH, pH7.5) were mixed followed by 95°C denaturation for 3 min. The reaction was slowly cooled down to anneal FragA1 and FragA2, and generate double-stranded Frag1. Double-stranded Frag2, Frag_me, and Frag_hemi were generated by annealing FragB1 and FragB2, Frag_mC1 and Frag_mC2, and Frag_C1 and Frag_mC2 (Supplementary Table S1), respectively. A ligation reaction was prepared with 1 μl of Frag1, 1 μl of Frag2, 1 μl of Frag_mC1 or Frag_mC2, 2 μl of 10× NEB T4 DNA ligase buffer, 1 μl of NEB T4 ligase (M0202L, NEB) and 14 μl of ddH₂O. This reaction was incubated at 20–25°C for 2 h, and the 146 bp ligation product was then purified by Ampure beads (A63880, Beckman). The ligation product was digested in a reaction containing 100 ng of this ligation product, 2 μl of 10x rCutSmart buffer (B6004S, NEB), 0.7 μl of 30× Enzyme Activator, 0.3 μl of MspJI (R0661S, NEB), and 15 μl of ddH₂O at 37°C for 2 h. The digestion products were then assessed using a 20% polyacrylamide gel.

The following items were key reagents and kits used in this study: GSK-3484862 (HY-135146, MedChemExpress), NEB T4 ligase (M0202L, NEB), MspJI (R0661S, NEB), EZ DNA Methylation-Lightning Kit (D5030, Zymo), anti-Rabbit IgG antibody (3418S, Cell Signaling Technology), anti-CTCF antibody (2729S, Cell Signaling Technology) and HiFi HotStart Uracil + DNA Polymerase (KK2801, Roche).