3C Analysis Protocol with BglII Digestion

For 3C analysis cells were crosslinked and digested as described for ChIP^{53 (link)}. Nuclei were resuspended in 500 μl of 1.2× NEB3 buffer (New England BioLabs) with 0.3% SDS and incubated at 37 °C for 1 h and then with 2% Triton X-100 for another 1 h. Digestion was performed with 800 U of BglII (New England BioLabs)overnight at 37 °C shaking. Digestion was checked loading digested and undigested controls on a 0.6% agarose gel. Then the sample was incubated with 1.6% SDS for 25 min at 65 °C and with 1.15× ligation buffer (New England BioLabs) and 1% Triton X-100 for 1 h at 37 °C. Ligation was performed with 1000 U of T4 DNA ligase (New England BioLabs) for 8 h at 16 °C and at 22°C for 30 min. DNA was purified with phenol-chloroform extraction after RNase A (Sigma) and Proteinase K (Sigma) digestion. As controls, BACs corresponding to the region of interested were digested with 100 U BglII in NEB3 buffer in 50 μl o/n at 37 °C. Then fragments were ligated with 400 U T4 DNA ligase overnight at 22°C in 40 μl. PCR products amplified with GoTaq Flexi (Promega) for BACs and samples were run on 2.5% agarose gels and quantified with ImageJ software. Primers are listed in Supplementary Table 3.