MNase-seq

MNase-seq was performed on unfixed cells as described [32] with the following modifications: For each time point, ~ 2 million cells were suspended in 10 mM HEPES pH 7.4, 0.5 mM PMSF, and 0.5% NP40 on ice in a 166-μL volume. MNase was added at a concentration of 2.5 U per million cells. The mixture was warmed to 37 °C before the addition of 3.5 μL 100 mM CaCl₂ (to 2 mM) to activate the MNase. Reactions were stopped by the addition of 170 μL of 2XSTOP (4.35 mL TM2, 340 μL 5 M NaCl, 200 μL 0.5 M EDTA, 100 μL 0.2 M EGTA, 25 μL RNase A, and 2 pg/mL yeast mononucleosomal spike-in DNA). After phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation, the DNA pellet was dissolved in 50 μL 1 mM Tris-HCl pH 8, 0.1 mM EDTA and used directly for 1.5% agarose gel analysis and for making sequencing libraries as described [32]. For yeast spike-in DNA, spheroplast nuclei [48] were digested with MNase down to mostly mononucleosomes and extracted with phenol-chloroform-isoamyl alcohol as described [16].