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MNase digestion

YZ Yanjun Zhang
DF Dong Fang
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A total of 4 × 106 cells were collected and washed in nuclear extraction buffer A (85 mM KCl, 10 mM Tris pH 7.5, 0.2 mM spermidine, 0.2 mM EDTA, 160 mM sucrose, and 250 μM PMSF) for 5 min. The samples were then lysed in nuclear extraction buffer B (buffer A + 0.1% NP40) for 5 min to extract nuclei. Nuclei were washed in digestion buffer (50 mM Tris pH 7.5, 20 mM KCl, 0.32 M sucrose, 4 mM MgCl2, and 3 mM CaCl2), and digested with micrococcal nuclease (2000 units/ml) for different time points: 0, 1, 2.5, 5, 10, and 20 min, at 37°. Equal volume of stop buffer (0.2% SDS and 10 mM EDTA) was added at 37° for 10 min. The samples were incubated with 10 μg/ml RNase A at 37° for 30 min, and then 100 μg/ml proteinase K at 55° for 1 h. DNA was purified with phenol/chloroform/isoamyl alcohol.

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