MNase-Seq of yeast:r4s12_rpb1-H1085Y_rep6
Nucleosomal DNAs were prepared by a method described elsewhere (van Bakel et al., 2013) with the following modifications. Yeast strains were grown in rich medium (YPD) to mid-log phase (~1.5X107/ml) and cross-linked with methanol-free formaldehyde (1% final concentration, Polysciences Inc) for 30 min and quenched with 0.25M final concentration of glycine (from 2.5M stock, pH=7). Cells were washed and digested with zymolyase-20T (Sunrise International) (6mg for 500ml culture) for ~17 min or until ~90% cells appeared as spheroplasts, followed by MNase (Thermo Fisher Scientific) digestion with different amount of MNase to generate less and more digested nucleosomes (in general, digests were limited such that at least mono, di, and trinucleosomes were still apparent in the lane in gel electrophoresis). Crosslinks on nucleosomes were reversed at 65 C in the presence of Proteinase K (G-Biosciences) overnight.