MNAse digestions

We cross‐linked 30 × 10⁶ exponentially growing cells by incubation for 5 min with 1% (v/v) freshly prepared formaldehyde (Thermo Fisher Scientific, #28908) at room temperature. Fixation was stopped by adding glycine to a final concentration of 0.125 M and incubating for 5 min at room temperature, with stirring. Cells were then washed three times in cold PBS, and their nuclei were extracted in 3 ml of lysis buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl₂, 0.2% Triton X‐100, 0.5 mM EGTA pH 8.0, 1 mM DTT, 1× protease inhibitor cocktail (Sigma, #P8340)) for 5 min at 4°C, centrifuged, and resuspended in digestion buffer (10 mM Tris–HCl pH 7.5, 10 mM NaCl, 3 mM MgCl₂, 1 mM CaCl₂, 1× protease inhibitor cocktail (Sigma, #P8340)). Micrococcal Nuclease (MNase; Thermo Fisher Scientific, #EN0181) digestions were performed for 15 min at 37°C, using either a final concentration of 10 U/ml for ChIp analyses or a series of four exponentially increasing concentrations of MNase (2.5, 10, 40 and 160 U/ml) for chromatin accessibility analyses. The reaction was stopped by adding 0.1 volumes of stop buffer (200 mM EDTA pH 8.0, 40 mM EGTA pH 8.0). For ChIp analyses, MNase digestion conditions were established so as to produce mostly mono‐ to hexa‐nucleosome fragments of 150–1,000 bp. Chromatin was then diluted by a factor of two in 2× complement buffer (40 mM Tris–HCl pH 8.0, 300 mM NaCl, 2% Triton X‐100) and sonicated 20 times for 30 s each, at 30 s intervals in the high mode at 4°C in a Bioruptor water bath sonicator (Diagenode). For chromatin accessibility analyses, the lowest MNAse concentration generated a mixture of oligo‐, di‐, and mono‐nucleosomes, whereas the highest concentration produced mostly mono‐nucleosomes (Appendix Fig S5A). After digestion, DNA molecules were extracted in phenol–chloroform and precipitated with ethanol and were then subjected to a size selection process with the SPRI‐select Reagent kit (Beckman Coulter #{"type":"entrez-nucleotide","attrs":{"text":"B23317","term_id":"2508948","term_text":"B23317"}}B23317) using a 0.5× ratio to remove DNA molecules of more than 1,000 bp in length (Appendix Fig S5B). The MNAse titration approach can be used to determine whether nucleosome release requires low or high levels of MNAse. Low levels of MNAse release large numbers of nucleosomes in accessible regions, leading to higher levels of DNA molecules. Nucleosomes embedded in less accessible regions are less likely to be released with such low levels of MNAse. Moreover, this approach provides additional information about the accessibility of loci. Indeed, highly inaccessible or condensed regions are not sensitive to higher concentrations of MNAse, whereas the chromatin of accessible regions is rapidly digested under the same conditions.