title: GSM3670739: L198-PBAT-dSMF; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Yeast cells in log phase (OD660 <1.0) were first centrifuged at 13,000 rpm for 1 minute, then washed with 100 uL 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 uL 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 30C at 300 rpm in a Thermomixer. The pellet was centrifuged for 2 minutes at 5,000 rpm, washed in 100 uL Sorbitol Buffer, and centrifuged again at 5,000 rpm for 2 minutes. Cells were then resuspended in 100 uL 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 4C, resuspended in 100 uL 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 4C. Finally, nuclei were resuspended in 100 uL 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 30C for 7.5 min. After this incubation, 128 pmol SAM and another 100 U of enzymes were added, and a further incubation at 30C 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 30C for 7.5 min. HMW DNA (~500 ng) was bisulfite converted using the EZ DNA Methylation-Lightning Kit (Zymo, Cat # D5030) by mixing 20 uL of purified DNA (~500 ng) with 130 uL DNA Methylation Lightning Conversion reagent and incubating at 98C for 8 minutes and then at 64C for 60 minutes. Bisulfite converted DNA was then cleaned up using the EZ DNA Methylation-Lightning Kit following manufacturer's instructions.  First strand synthesis was carried out by mixing 20 uL bisulfite converted DNA, 19.75 uL H2O, 5 uL 10X Blue Buffer (ThermoFisher), 1.25 uL 10 mM dNTP (NEB), and 4 uL custom-designed biotinylated adapter. Samples were then incubated at 94C for 5 minutes, and at 4C for 5 minutes, after which 1.5 uL Klenow (3' -> 5' exo minus; MCLab) were added, and the reaction was incubated at 4C for 15 minutes, at 37C for 90 minutes, and at 70C for 5 minutes. First-strand reaction cleanup was carried out using 50 uL AMPure XP beads (Beckman Coulter); DNA was eluted 50 uL EB buffer.  Biotinylated DNA was captured on streptavidin beads. A total of 20 uL streptavidin Dybaneads M-280 (ThermoFisher) per sample were added to a PCR tube, separated on a magnet and then resuspended in 50 uL 2X BW(Li) buffer (6.3 g LiCl, 0.5 mL Tris-HCL pH 8.0, and 0.1 mL 500 mM EDTA for 50 mL total volume), to which the 50 uL of eluted first-strand reaction DNA was added. Beads were then incubated at room temperature for 30 minutes, washed with 180 uL 2X BW(Li) buffer, twice with 0.1 N NaOH (by resuspending well and incubating at room temperature for 2 minutes), washed again with 180 uL 2X BW(Li) buffer, then with 180 uL 10 mM Tris-HCL pH 7.5.  Second-strand synthesis was carried out by resuspending streptavidin beads in the following reaction mix: 5 uL 10 X Blue Buffer, 1.25 uL 10 mM dNTPs, 39.75 uL H2O, 4 uL custom-designed second-strand adapter. Samples were then incubated at $94C for 5 minutes, and at 4C for 5 minutes, after which 1.5 uL Klenow (3' -> 5' exo minus) were added, followed by further incubation at 4C for 15 minutes, at 37C for 30 minutes, and at 70C for 5 minutes. Beads were separated on magnet and the chase reaction was carried out by resuspending in a mix of 5 uL 10X Thermo Pol Buffer, 1.25 uL 10 mM dNTPs, 43.5  uL H2O, and 1  uL Bst DNA Polymerase Large Fragment (NEB). Samples were incubated at 65C for 30 minutes, then again separated on magnet.
attributes.tag: GEO Accession
attributes.value: GSM3670739