Chromatin and Transcriptome Profiling of Metarhizium

First, 2.5 × 10⁵ spore/mL YPD cultures were grown for 16 h at 26 °C and 250 rpm in duplicates. Then, the same culture was utilized to obtain ChIP and RNA samples to ensure minimal experimental differences. ChIP-grade antibodies α-H3K9me2 (ab1220, Abcam), α-H3K9me3 (ab8898, Abcam), and α-RNA pol II (39497, Active Motif) were utilized to immunoprecipitate chromatin-bound DNA, following a previously established procedure (40 (link)). Libraries were prepared using Roche KAPA HyperPrep Kits and sequenced with Illumina NovaSeq 6000 sequencing system for 100-bp paired-end reads. Total RNA was purified using a QIAGEN miRNeasy Mini Kit, and RNA samples were divided into small and long RNA preparations. rRNA-depleted RNA libraries (long RNA) were prepared using Illumina Stranded Total RNA Prep with the Ribo-Zero Gold rRNA Removal Kit and M. lusitanicus rDNA-specific probes, and cDNA sequenced in a NovaSeq 6000 sequencing system for 150-bp paired-end reads. Last, sRNA libraries were amplified using the QIAseq miRNA library kit, and sequenced in an Illumina NextSeq 500 High-Output sequencing system to obtain 75-bp single reads.