title: GSM4193308: WT3_BamHI-HF_800U_30min_rep1; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Nuclei from ca. 0.1 g wet cell pellet were pre-washed in methylation buffer, centrifuged and resuspended either in 400 µl 1x CutSmart buffer (NEB) for BamHI-HF and AluI or in 1x NEB2.1 for HindIII. Digestions were started by RE addition, incubated for 30 or 120 minutes at 37°C and stopped by 10 mM EDTA and 0.5% SDS (final conc.). DNA preparation was as above. For the cut-all cut method, Schizosaccharomyces pombe gDNA, fully digested with the corresponding RE, was spiked in at 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 (final conc.). 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: GSM4193308