title: Diamide time course
design: Yeast cells in log phase (OD660  1.0) were first centrifuged at 13,000 rpm for 1 minute, then washed with 100 L Sorbitol Buffer(1.4 M Sorbitol, 40 mM HEPES-KOH pH 7.5, 0.5 mM MgCl2), and centrifuged again at 13,000 rpm for 1 minute. Cells were then spheroplasted by resuspending in 200 L Sorbitol Buffer with DTT added at a final concentration of 10 mM and 0.5 mg/mL 100T Zymolase, followed by incubating for 5 minutes at 30 C at 300 rpm in a Thermomixer. The pellet was centrifuged for 2 minutes at 5,000 rpm, washed in 100 L Sorbitol Buffer, and centrifuged again at 5,000 rpm for 2 minutes. Cells were then resuspended in 100 L ice-cold Nuclei Lysis Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, 0.5% NP-40) and incubated on ice for 10 minutes. Nuclei were then centrifuged at 5000 rpm for 5 min at 4 C, resuspended in 100 L cold Nuclei Wash Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA), and centrifuged again at 5,000 rpm for 5 min at 4 C. Finally, nuclei were resuspended in 100 L M.CviPI Reaction Buffer (50 mM Tris-HCl pH 8.5, 50 mM NaCl, 10 mM DTT). Nuclei were then first treated with M.CviPI + EcoGII by adding 200 U of M.CviPI (NEB) and 200 units of EcoGII (NEB), SAM at 0.6 mM and sucrose at 300 mM, and incubating at 30 C for 7.5 min. After this incubation, 128 pmol SAM and another 100 U of enzymes were added, and a further incubation at 30 C for 7.5 min was carried out. Immediately after, M.SssI treatment followed, by adding 60 U of M.SssI (NEB), 128 pmol SAM, MgCl2 at 10 mM and incubation at 30 C for 7.5 min. The reaction was stopped by adding an equal volume of Stop Buffer (20 mM Tris-HCl pH 8.5, 600 mM NaCl, 1% SDS, 10 mM EDTA)
identifiers.namespace: SUB6542771
identifiers.id: diamide_60_min_rep1