GSM9369597: BY4741_wildtype_MNase_replicate_1; Saccharomyces cerevisiae; MNase-Seq
BY4741 yeast cultures were first crosslinked with 1% formaldehyde for 15 minutes at room temperature. 125mM Glycine was added to quench the reaction. Cell pellets were resuspended in spheroplasting buffer (1M Sorbitol, 5 mM fi-mercaptoethanol, 50mM Tris pH 7.5, 2 mg/ml zymolyase (AMS Bio cat no: 120493-1); 1 mL of buffer per 20 mL of cell culture) and incubated for 15 min. at room temperature. The derived spheroplasts were treated with 100U MNase (NEB cat no: M0247S) for 30-40 min at 37°C. The reactions were stopped by the addition of EDTA pH 8.0 (50 mM final conc.) and EGTA pH 8.0 (50 mM final conc.). Samples were then incubated with RNase A (Thermo Scientific, EN0531, final conc. 0.2 mg/ml) at 42°C for 30 min to digest RNA. The crosslinks were reversed by adding SDS and proteinase K (Invitrogen cat no: 25530049, 1mg/ml final concentration) and incubation at 65°C for 45 min. DNA was extracted using the Monarch PCR & DNA cleanup kit. Samples were resolved on 1% agarose gel to evaluate the digestion. Mononucleosome-sized bands were extracted and libraries were constructed from 10ng purified DNA using the Watchmaker Genomics according to the manufacturer's instructions. Paired-end sequencing was performed on AVITI (2x 75bp cycles).