GSM7591788: INO80_ORC_foldamer_14_rep1; 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 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 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, Chacin et al., 2023) by using 10-30 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 using the 4150 TapeStation System (Agilent). The pool was sequenced on an Illumina NextSeq1000 in 60 bp paired-end mode (Laboratory for Functional Genome Analysis, LAFUGA, LMU Munich).