Chromatin Conformation Capture (3C) Assay

3C was adapted from a previously published protocol^{43 (link)}. Yeast strains were grown in 200 mL synthetic media without methionine to OD660 = 0.3. Cells were fixed with 3% formaldehyde (Sigma, 252549) for 20 min at 25 °C and then quenched with glycine for 20 min at room temperature. Cells were collected by centrifugation and washed with the same medium. Cell pellets were resuspended in 1 mL TBS, 1% Triton X-100 and 1X protease inhibitor cocktail (Thermo Fisher, 78420). Cell lysis was performed by adding 500 μL acid-washed glass beads (Sigma, G8772) and vortexing for 25 min at 4 °C. The chromatin was recovered through centrifugation, washed with 1 mL TBS, resuspended in 500 μL 10 mM Tris-HCl buffer and digested with DpnII (NEB, R0543) overnight at 37 °C. The digested DNA fragments were ligated by T4 DNA ligase (Thermo Fisher, EL0014) for 4 h at 16 °C. Crosslink was reversed by incubation with proteinase K at 65 °C overnight. DNA was purified by the standard phenol-chloroform extraction. PCR primers (Supplementary Table 2) were designed for the regions of interest near DpnII cutting sites, and PCR reactions were performed for testing interactions between these regions under different conditions with 4 ng input DNA and 45 amplification cycles.