title: GSM3854453: combined ORE and ODM-seq map; Saccharomyces cerevisiae; OTHER
library_construction_protocol: DNA methylation in chromatin: Yeast nuclei were washed in methylation buffer (20 mM HEPES-NaOH pH 7.5, 70 mM NaCl, 0.25 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.5% (v/v) glycerol, 1 mM DTT, 0.25 mM PMSF (Darst et al. 2012)). Per reaction, roughly 0.1 g nuclei pellet was resuspended in 800 µl methylation buffer containing 640 µM freshly added SAM (S-adenosylmethionine). For CpG methylation 200 U M.SssI (NEB) were added; for GpC methylation 200 U C.CviPI (NEB) and additionally 10 mM DTT were freshly added to the reaction. Methylation reactions were transferred to Slide-A-Lyzer MINI Dialysis Devices 10 K MWCO (ThermoFisher Scientific) and incubated while dialyzing against 15 ml methylation buffer containing 200 µM freshly added SAM at 25°C for CpG or 37°C for GpC methylation. To monitor completeness of methylation, 1 µg fully assembled pUC19-601-25mer plasmid (pFMP233) (Lieleg et al. 2015, Lowary and Widom 1998) was spiked into the methylation reaction. Reactions were stopped by addition of 0.5% SDS (final concentration) followed by reverse crosslinking, if necessary. The DNA was deproteinized by protease K digestion, phenol-chloroform extracted and RNase A digested.   Restriction enzyme digestion of chromatin: 0.1 g nuclei pellet per reaction was pre-washed in methylation buffer, centrifuged again and then resuspended either in 400 µl 1x CutSmart buffer (NEB) for BamHI-HF and AluI digests or in 1x NEB2.1 for HindIII digests. Digestion reactions were started by RE addition (Supplementary Table 3) and incubated for 30 or 120 minutes at 37°C. Reactions were stopped by 10 mM EDTA and 0.5% SDS (final concentration) followed by DNA extraction as described above. For the cut-all cut method, S. pombe genomic DNA (gDNA), fully digested with the corresponding RE, was spiked into the sample (5-10% DNA mass of the final sample) before phenol-chloroform extraction. After RNase A digestion, half of the sample was digested for a second time with 100 U of the corresponding RE and in the corresponding buffer for 1.5 h at 37°C. The reaction was stopped by 15 mM EDTA. Illumina sequencing library construction and sequencing  Purified DNA was sheared to ~150 bp fragments with the Covaris S220 and concentrated using the NucleoSpin Gel and PCR Clean-up Kit (Macherey&Nagel). 0.5 – 1 µg as determined by Qubit (ThermoFisher Scientific) was used for library preparation. First, DNA was end polished (15 U T4 DNA Polymerase, 50 U T4 PNK, 5 U Klenow (NEB)) for 30 minutes at 20°C, purified with AmPure XP beads (Beckman Coulter), then subjected to A-tailing (15 U Klenow exo-(NEB)) for 30 minutes at 37°C and purified again. For adapter ligation 15 U T4 DNA Ligase (NEB) and 75 to 150 pmol NEBNext Adaptor or NEBNext Methylated Adaptor for bisulfite conversion were added. The reaction was incubated for 20 minutes at 25°C followed again by AmPure XP bead purification. Half of the end-polished and adapter ligated sample was subjected to 8 cycles of PCR using the NEBNext Multiplex Primers for Illumina and Phusion Polymerase (NEB). Methylated samples were bisulfite converted following the manufacturer's instructions of the Qiagen EpiTect Bisulfite Kit. 3 µl of the converted sample was subjected to 12-14 cycles of PCR using the NEBNext Multiplex Primers for Illumina and Phusion U Polymerase (ThermoFisher Scientific). All samples were sequenced by LaFuGa (Illumina HiSeq 1500, 50 bp paired-end mode).
attributes.tag: GEO Accession
attributes.value: GSM3854453