Biotin Labeling of DNA Contacts

Sample beads were washed in 200 μl of ChIRP elution buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 3 mM Mgcl₂, 10 mM dithiothreitol, 0.1% NP-40) and then resuspended in 200 μl of ChIRP elution buffer containing 4 μl of RNase A (5 mg ml⁻¹, Thermo) and 4 μl of RNase H (5 U μl⁻¹, Thermo)^{24 (link)}. Samples were then incubated at 37 °C with shaking for 30 min, placed on a magnet and eluted into new tubes. Elution was repeated with another 200 μl of ChIRP elution buffer containing RNases. SDS to a final concentration of 0.5% and 20 μl of Proteinase K (20 mg ml⁻¹, Thermo Fisher) were then added to the 400-μl reaction. Samples were incubated at 55 °C for 45 min with shaking. Samples were purified with DNA Clean and Concentrator columns (Zymo Research) and eluted in 50 μl of water. To incorporate a biotin into the DNA contacts for capture, we carried out copper-free CLICK chemistry using 1 μl of DIBO-Biotin (Thermo) added to the sample and incubating at 37 °C for 1 h with shaking. Samples were purified again with DNA Clean and Concentrator columns (Zymo Research) and eluted in 10 μl of water.