Trimethylated h3 lys9

ChIP assay was performed using the ChIP-IT Express kit (Active Motif, Carlsbad, USA) following the manufacturer's protocol as described previously (Pham, et al. 2015 ). Fungal mycelia were grown in CM liquid for 4 days at 26° on an orbital shaker (120 rpm). A portion of mycelia (50 mg) was harvested and incubated at room temperature for 15 min in 10 ml of phosphate-buffered saline (PBS) containing formaldehyde at a concentration of 1%. Chromatin was sheared by sonication using a Bioruptor apparatus (Cosmo Bio Co., Ltd., Japan) for three cycles of 1 min on at high intensity (200 W) and 30 s off, followed by five cycles of 1 min on at medium intensity (160 W) and 30 s off. The size of the sheared chromatin was around 100 to 500 bp as determined by agarose gel electrophoresis. Antibodies against demethylated H3 Lys 4 (Active Motif, #39141), trimethylated H3 Lys9 (Active Motif, #39161), and trimethylated H3 Lys27 (Active Motif, #39156) were obtained from Active Motif. ChIPed DNA was recovered by phenol-chloroform extraction and ethanol precipitation. Libraries for high-throughput sequencing were constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina and sequenced using HiSeq X at Macrogen Japan Co. Ltd (Kyoto, Japan).

ChIP assay was performed using the ChIP-IT Express kit (Active Motif, Carlsbad, USA) following the manufacturer's protocol as described previously (Pham, et al. 2015 ). Fungal mycelia were grown in CM liquid for 4 days at 26° on an orbital shaker (120 rpm). A portion of mycelia (50 mg) was harvested and incubated at room temperature for 15 min in 10 ml of phosphate-buffered saline (PBS) containing formaldehyde at a concentration of 1%. Chromatin was sheared by sonication using a Bioruptor apparatus (Cosmo Bio Co., Ltd., Japan) for three cycles of 1 min on at high intensity (200 W) and 30 s off, followed by five cycles of 1 min on at medium intensity (160 W) and 30 s off. The size of the sheared chromatin was around 100 to 500 bp as determined by agarose gel electrophoresis. Antibodies against demethylated H3 Lys 4 (Active Motif, #39141), trimethylated H3 Lys9 (Active Motif, #39161), and trimethylated H3 Lys27 (Active Motif, #39156) were obtained from Active Motif. ChIPed DNA was recovered by phenol-chloroform extraction and ethanol precipitation. Libraries for high-throughput sequencing were constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina and sequenced using HiSeq X at Macrogen Japan Co. Ltd (Kyoto, Japan).