We extracted mononucleosomes using a micrococcal nuclease (MNase) digestion protocol that was described previously [47, 85]. Approximately 90 OD600 units of cells were crosslinked at 30 °C for 20 min with formaldehyde (1% v/v) and the reaction was quenched with glycine (125 mM). Subsequently, cells were resuspended in 20 ml of buffer Z (1 M sorbitol, 50 mM Tris-HCl pH 7.4) plus β-mercaptoethanol (10 mM) and treated with 250 μg of T100 Zymolase (MP Biomedicals) at 30 °C for 60 min. Next, spheroplasted cells were pelleted (4000g, 10 min) and resuspended in 1 ml NP buffer (0.5 mM spermidine, 1 mM β-mercaptoethanol (β-ME), 0.075% (w/v) Tergitol solution-type NP-40 detergent (NP-40), 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2). Separate aliquots of this lysate were treated with 400, 167, and 80 gel units of MNase (M0247S, NEB) for 30 min at 37 °C, and the reaction was quenched with EDTA (10 mM). In total, 100 μl of MNase treated and untreated extracts were reverse crosslinked at 65 °C overnight in 1 ml of SDS-TE (1.0% w/v SDS, 10 mM Tris pH 8.0, 1 mM EDTA, 4.5% v/v proteinase K). Extracts were then treated with 20 μl RNase A (10 mg/ml), DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated at − 20 °C overnight in ethanol with 0.3 M sodium acetate. To check the extent of MNase digestion, purified DNA fragments were electrophoresed on a 2% agarose gel. The extracts which showed a mostly mono-nucleosome pattern were gel purified (Macherey-Nagel) and used as inputs for the KAPA Hyper Prep Kit (KK8504, Roche). Ligations were done using KAPA single indexed adapters Set A (KK8701, Roche) or Set B (KK8702, Roche). Libraries were constructed according to the manufacturer’s instructions. Purified libraries were sequenced on an Illumina HiSeq 2500 (100 bases paired-end reads and approximately 8 million reads per library).

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