GSM4879812: MNase-seq: spt6-YW spt5-QS, 30C, 2; Saccharomyces cerevisiae; MNase-Seq
DOI: 10.1016/j.molcel.2018.09.005 At a density of approximately 1 × 107 cells/ml (OD600 = 0.5), cells were crosslinked using 2% formaldehyde for 30 minutes and then treated for 10 minutes with 125 mM glycine before collecting an equal number of cells for each strain. The cells were resuspended in 40 mL of sorbitol buffer (1 M sorbitol, 50 mM Tris pH 7.4, 10 mM ?-mercaptoethanol) and incubated for 30 minutes at 30°C with 10 mg of zymolase 100T (US Biological) per gram of cells. Spheroplasting efficiency was assessed by microscopy and was more than 95% of total cells. The spheroplasts were collected and resuspended in NP buffer (1 M sorbitol, 50 mM sodium chloride, 10 mM Tris pH 7.4, 5 mM magnesium chloride, 1 mM calcium chloride, 0.075% NP-40, 1 mM ?-mercaptoethanol, 500 ?M spermidine). Micrococcal nuclease (MNase; Sigma) was dissolved in Ex50 buffer (10 mM HEPES pH 7.6, 50 mM sodium chloride, 1.5 mM magnesium chloride, 0.5 mM EGTA, 10% glycerol, 1mM dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride) prepared to produce 500 units per 840 ?l stock as recommended by the manufacturer. The spheroplasts were divided into aliquots and incubated for 20 minutes at 37°C with increasing amounts of MNase, ranging from 2 to 15 ?L of the stock. Digestion was stopped by addition of stop buffer (5% SDS, 100 mM EDTA), samples were subjected to proteinase K digestion and reverse-crosslinking at 65°C overnight, followed by DNA purification. The efficiency of MNase digestion was quantified using DNA fragment size analysis (Agilent Bioanalyzer) to establish an MNase titration curve for each strain. The MNase concentrations which yielded approximately 80% mononucleosomal DNA were selected for library construction. The samples were mixed with the MNase-digested spike-in DNA from S. pombe (strain 972) based on the original cell count (100 ng of spike-in DNA per 7 X 107 S. cerevisiae cells). Mononucleosomal DNA was purified using size-selected gel extraction.