Bioruptor apparatus

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^TM Express kit (Active Motif, Carlsbad, USA) following the manufacturer’s protocol. Fungal mycelia were grown in CM liquid media for 4 days at 26 ˚C 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 3 cycles of 1 min on at high intensity (200 W) and 30 s off, followed by 4 cycles of 1 min on at medium intensity (160 W) and 30 s off. The size of the sheared chromatin was around 200– 1000 bp as determined by agarose gel electrophoresis. Antibodies against trimethylated H3 Lys9 (Active Motif, #39161) and trimethylated H3 Lys27 (Active Motif, #39156) were obtained from Active Motif. ChIP-enriched genomic DNA fragments were subjected to qPCR analysis using the primers for MAGGY (pair B, see Supplementary Data 1 and above) and the actin gene (Supplementary Data 1). Input DNA was used to normalize the data. To calculate relative enrichment, the values for MAGGY sequences were divided by the values for the actin gene.

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).