GSM4826110: spt4del_MNase-seq_rep1; Saccharomyces cerevisiae; MNase-Seq
Cells were harvested and incubated in 2.8 mM EDTA, pH 8, 0.7 M 2-mercaptoethanol (30°C, 30 min). Then samples were pelleted and washed with 1 M sorbitol. The pellet was resuspended in 1 M sorbitol, 5 mM 2-mercaptoethanol solution and zymolase solution (30°C for 30 min). The lysate was pelleted and washed with 1 M sorbitol. Nuclei were resuspended in 18% Ficoll, 20 mM KH2PO4, 1 mM MgCl2, 0.25 mM EGTA, 0.25 mM EDTA and stored as pellet. Nuclei was digested with 80U of MNase (Nuclease S7; 37°C, exactly 10 min). Reaction was quenched with 5% SDS, 50 mM EDTA solution (65°C). Following protein and RNA digestion, DNA was eluted with Zymoclean ChIP concentrator kit according to the manufacturer's instructions. Isolated DNA was run on a 1.5% agarose-TBE gel and the mononucleosomal DNA band was gel extracted. standard NEBNext Ultra II DNA Library Prep Kit for Illumina