GSM9290595: Ies6, clone 1; Saccharomyces cerevisiae; MNase-Seq
Cells were grown to OD600 0.8 in 500 ml YPAD media. Spheroplasts were prepared by treating cells with 2 mg zymolyase at 30 °C for 20 min, followed by washing with ice-cold 1 M sorbitol. Nuclei were resuspended in MNase digestion buffer (15 mM Tris–Cl pH 7.5, 50 mM NaCl, 1.4 mM CaCl2, 0.2 mM EGTA pH 8.0, 0.2 mM EDTA pH 8.0, 5 mM β-mercaptoethanol) and digested with increasing amounts of MNase for 20 min at 37 °C. DNA was purified via Proteinase K treatment, followed by Phenol: Chloroform: Isoamyl alcohol extraction and RNase digestion. Samples with ~70% mononucleosome content were selected for library preparation. Sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep kit for Illumina and sequenced in 50 bp paired-end mode, yielding five to ten million reads per sample.