RNA-seq Analysis of MoLID2 (MoKDM5)-dependent Genes in Magnaporthe oryzae 70-15

Purpose: The goals of this study are to find out the genes regulated directly or indirectly by a trimethyl H3K4 histone demethylase Molid2 (MoKdm5) by RNA-seq in the rice blast fungus Methods: Mycelial mRNAs of the wild type strain 70-15, ΔMolid2 incubated in H2O at 25˚C for 4 h, in triplicate independently for each strain, were extracted and isolated by RNeasy Plant mini kit (QIAGEN) and AMPure XP beads (Beckman). RNA-sequencing libraries were constructed using NEBNext RNA sample preparation kit (NEB). Samples were sequenced in a 1 x 100 nt way on Illumina Hiseq2500 instrument using TruSeq PE Cluster Kit v3 - cBot - HS (Illumina) and TruSeq SBS Kit v3-HS (Illumina).The M. oryzae genome database (MG8) (www.broadinstitute.org) was used as a reference gene database. Genome-wide transcript levels of genes were quantified in fragments per kilobase of exon model per million mapped reads (FPKM) (Trapnell 2010). Gene Ontology (GO) enrichment analyses for differentially expressed genes were performed using hypergeometric test with topGO, and p-values were adjusted with Bonferroni for multiple testing (Alexa 2006). And we selected FDR < 0.05 as enrichment terms. Results and Conclusions: We identified 3400 genes regulated by Molid2 through RNA-seq. Mycelial mRNA profiles of wild type strain 70-15 (WT), ΔMolid2 incubated in H2O for 4 h were generated, in biologic triplicate, by Illumina Hiseq2500 in a 1 x 100 nt way