Micro‐capture‐C

Micro‐capture‐C was performed as previously described (Hua et al, 2021). Cells were fixed with formaldehyde (2% (wt/vol)) for 10 min at room temperature and permeabilized with digitonin (0.005% (wt/vol)). 2–3 × 10⁶ cells were treated with MNase (NEB M0247) ranging from 10 to 30 Kunitz U in 800 μl of custom buffer (Tris–HCl pH 7.5 10 mM, CaCl₂ 1 mM) for 1 h at 37°C. Ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA) 5 mM (Sigma E3889) was added to quench the reaction. The reaction was centrifuged (5 min, 300 × g) and the supernatant discarded. The cells were resuspended in 1 ml phosphate buffered saline with 5 mM EGTA of which 200 μl was used to measure the digestion efficiency. The remainder was centrifuged (5 min, 300 × g) and the supernatant discarded. Cells were resuspended in DNA ligase buffer (Thermo Scientific EL0013) supplemented with dNTPs (dATP, dCTP, dGTP and dTTP; 400 μM final concentration of each (Thermo Fischer R0191)); EGTA 5 mM; T4 Polynucleotide kinase PNK 200 U/ml (NEB M0201L); DNA Polymerase I Large (Klenow) Fragment 100 U/ml (NEB M0210L) and T4 DNA ligase 300 U/ml (Thermo Scientific EL0013). The reaction was incubated at 37°C for 2 h followed by 20°C for 8 h using an Eppendorf Thermomixer at 500 rpm. The ligation reaction was centrifuged and the supernatant was discarded. The chromatin was decrosslinked with proteinase K at 65°C (> 2 h) and the DNA was extracted. Double oligonucleotide capture was performed as previously described (Davies et al, 2015). Data were analyzed with a custom analysis pipeline specifically developed for MCC data analysis.