Native ChIP for Leptosphaeria maculans

We first attempted to adapt the ChIP protocol developed for N. crassa and Fusarium species [28] (link), [43] (link), but found that sonication or bead-beating and formaldehyde crosslinking resulted in very poor shearing or yields of precipitated DNA. We thus subjected L. maculans to “native ChIP” (no crosslinking) and isolated mono- and dinucleosomes after digesting ∼300 mg of mycelium per sample with microccocal nuclease (MNase) for 25 min at 37°C (L.R. Connolly and M. Freitag, unpublished data). Input (40 µl of the whole cell lysate) was stored (−20°C) for each sample and used to normalise data from qPCR and qualitative PCR. For immunoprecipitation, each sample was split into two replicates of 250 µl lysate each and 3.5 µl H3K9me3 antibody (Active Motif 39161) was added for a total of two replicates per sample. Precipitations with H3K4me2 antibody (Millipore 07-030) were done in parallel as controls for ChIP efficiency. Because we used a native ChIP protocol, yields were consistently lower than from ChIP experiments with crosslinked chromatin.