Novel insights into the role of the histone variant H2A.Z in the plant pathogen Fusarium fujikuroi [Mnase-seq]
Localization of nucleosomes in F. fujikuroi Overall design: Fungal mycelia were cultivated for three days in liquid ICI (6 mm glutamine) prior crosslinking with 1% formaldehyde. Crosslinked and quick-frozen mycelia (3 days, liquid synthetic ICI, 6 mM glutamine) was treated with MNaseI (60 U/µL, Thermo Fisher Scientific) for 4 minutes. The reaction was stopped by the addition of 0.5 M EDTA (final conc. 40 mM EDTA). Reverse cross-linking of digested DNA was performed for at least 30 minutes at 65 °C. Subsequently,  samples were diluted and treated with proteinase K (1h and 40°C, Thermo Fisher Scientific) for protein degradation. DNA fragments were extracted using phenol and separated on an agarose gel to check for successful MNase digestion. Fragmented DNA was cut from the gel at 150 bp and extracted using the Monarch® DNA Gel Extraction Kit (NEB®). Quality control, library preparation and sequencing were performed at the Vienna BioCenter Core Facilities (Vienna, Austria).