H3k9me3 antibody

OVCAR3-KRAB cells were transfected with sgRNAs targeting either scramble1 (non-targeting) or SE60 (2 pooled sgRNAs) following the same protocol mentioned above for “CRISPRi for SE14 and SE60.” For each of the two replicates conducted per condition, 1–2 million cells were used for fixation with 11% formaldehyde following Active Motif’s Epigenetic Services ChIP Fixation Protocol. ChIP-seq for H3K9me3 was performed by Active Motif using H3K9me3 antibody (Active Motif, 39161) with spike-in Drosophila normalization. ChIP-seq libraries underwent 75 bp single-end sequencing on an Illumina NextSeq 5000 instrument by Active Motif.

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∼300 mg of mycelium per sample with microccocal nuclease (MNase) for 25 min at 37°C (L.R. Connolly and M. Freitag, unpublished data).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.

For ULI-NChIP-seq, 10 000 or 40 000 cells were used per reaction. The ULI-NChIP procedure was performed as previously described (17 (link),18 ). Briefly, 2 μg of H3K27ac antibody (39133, Active Motif), H3K4me1 antibody (39297, Active Motif), H3K4me3 antibody (9727, Cell Signaling Technology), H3K27me3 antibody (9733, Cell Signaling Technology) or H3K9me3 antibody (39161, Active Motif) was used for each immunoprecipitation reaction. The sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645, New England Biolabs) following the manufacturer's instructions. The CUT&Tag assays for CTCF were performed using 50 000 sorted ESCs and 2CLCs as previously described (19 (link)). Two or three replicates were performed. Barcoded libraries were pooled and sequenced on an Illumina HiSeq X Ten platform in the paired-end mode.