Ecori buffer

3C assays were carried out essentially as described previously [50 (link)]. Briefly, cells were fixed with 2% formaldehyde and lysed in 10 mM Tris pH8, 10 mM NaCl, 0.2% NP-40. Nuclei were resuspended in EcoRI buffer (New England Biolabs) containing 0.3% SDS for 1 h at 37 °C. After addition of 2% Triton X-100 for 1 h at 37 °C, nuclei were digested overnight with 400U of EcoRI-HF (New England Biolabs). Samples were incubated with 1.6% SDS at 65 °C, diluted tenfold with 1.15× ligase buffer (Roche) and incubated with 1% Triton X-100 at 37 °C. Ligation was performed for 4 h at 16 °C with 100U of T4 DNA ligase (Roche). After de-cross-linking and RNA removal, DNA was purified and analyzed by PCR using unidirectional primers designed to amplify across ligation junctions. Positive control PCR were carried out using genomic PCR fragments covering the restriction sites of interest that were then digested and ligated.

EcoRI (New England BioLabs Inc. Cat#R0101S) activity in perchlorate solutions generally consisted of 0.05 U/µL enzyme, 1X EcoRI buffer (New England BioLabs Inc. Cat#B7006S), and 1 µM duplex DNA (see Supplementary Table 1). NaX concentration varied per experiment as noted in the text. Reactions were started by incubating at 37 °C. Reactions were stopped and cleaned up with the addition of 75% ethanol. DNA cleavage was quantified using a 5′-fluorescein label and a 20% urea-PAGE gel.
RNase HII (New England BioLabs Inc. Cat#M0288S) was assessed with the same protocol but in 1X ThermoPol Buffer (New England BioLabs Inc. Cat#B9004S).
TaqI-v2 ((New England BioLabs Inc. Cat#R0149S) was assessed with the same protocol but in 1X rCutSmart Buffer (New England BioLabs Inc. Cat#B6004S) and incubated at 65 °C. DNA was resolved on a 10% urea-PAGE gel.
All gels were analyzed using an Omega Lum G Imaging System using the Omega Lum Image Capture Software V 2.1.2017.0. GelQuant (GelQuant.NET V 1.8.2) was used for quantification.