GSM6383598: isw1Δ; Saccharomyces cerevisiae; MNase-Seq
The nuclei samples were digested with 16-32 U MNase in MNase buffer (150 mM Tris-HCl pH 7.5, 500 mM NaCl, 14 mM CaCl2, 2 mM EDTA, 2 mM EGTA and 50 mM β-mercaptoethanol) to get an 80 % mono- / 20 % di- nucleosome distribution. The reaction was stopped by addition of 10 mM EDTA and 0.5 % SDS. This was followed by a proteinase K digestion, phenol-chloroform extraction, ethanol precipitation, RNAse A digestion and isopropanol precipitation. The purified DNA was run in 1.5 % agarose gels for 1.5 h at 110 V constant in 1X TAE, the mononucleosomal DNA was cut out and purified using the PureLink Quick Gel Extraction kit (Invitrogen). The libraries from in vivo samples were prepared with the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB). The amplified DNA was run in a 1.5 % agarose gel, cut out, purified and the concentration was measured with the Qubit dsDNA HS Assay Kit (Fisher Scientific). The libraries were diluted to 10 nM, pooled according to the sequencing reads (~ 5 million reads/sample), and quantified via BioAnalyzer (Agilent). The pool was sequenced either on an Illumina HiSeq 1500 in 50 bp single-end mode or on an Illumina NextSeq 1000 in 60 bp paired-end mode (Laboratory for Functional Genome Analysis, LAFUGA, LMU Munich).