GSM6383475: SGD_Fun30_ORC_rep3; Saccharomyces cerevisiae; MNase-Seq
The reactions with reconstituted chromatin were incubated with 100 U MNase (Sigma-Aldrich) and 1.5 mM CaCl2 for 5 min at 30 °C. The digest was stopped by the addition of 10 mM EDTA and 0.5 % SDS. This was followed by a proteinase K treatment for 1 h at 37 °C and ethanol precipitation. The sequencing libraries of the vitro reconstituted chromatin samples were prepared as previously described (Oberbeckmann, E., Niebauer, V. et al., 2021; Oberbeckmann, E., Krietenstein, N. et al., 2021) by using 10-50 ng mononucleosomal DNA. 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).