Total RNA was extracted from mycelia grown for 3 days in liquid ICI media (containing either 6 or 60 mM glutamine) and from infected rice seedlings after 7 dpi using TRIzol Reagent (Life Technology, Karlsruhe, Germany) and purified using an RNeasy Plant MinElute Cleanup Kit (Qiagen, Hilden, Germany). The quality of DNase-treated RNA (28S:18S > 1.0; RIN≥ 6.5; OD260/280 ≥1.8; OD260/230 ≥ 1.8) was determined using an Agilent Bioanalyzer. The high quality RNA was sent to BGI Tech Solutions Co., Limited (Hong Kong) for library construction and sequencing by Illumina HiSeq2000 technology.
RNA-seq reads were mapped on the reference genome using tophat2 (v2.0.8). The interval for allowed intron lengths was set to min 20 nt and max 1 kb [84 –86 (link)]. We used cufflinks to determine the abundance of transcripts in FPKM (fragments per kilobase of exon per million fragments mapped) and calculated differentially expressed genes using cuffdiff [85 (link),86 (link)]. The gene models were included as raw junctions. Genes with a minimum of fourfold increase or decrease in expression (|log2 of the FPKM values +1| ≥ 2) between the two experimental conditions were considered as regulated. The RNA-seq data has been deposited in NCBI's Gene Expression Omnibus [87 (link)] and are accessible through GEO Series accession number GSE89480.
RNA-seq reads were mapped on the reference genome using tophat2 (v2.0.8). The interval for allowed intron lengths was set to min 20 nt and max 1 kb [84 –86 (link)]. We used cufflinks to determine the abundance of transcripts in FPKM (fragments per kilobase of exon per million fragments mapped) and calculated differentially expressed genes using cuffdiff [85 (link),86 (link)]. The gene models were included as raw junctions. Genes with a minimum of fourfold increase or decrease in expression (|log2 of the FPKM values +1| ≥ 2) between the two experimental conditions were considered as regulated. The RNA-seq data has been deposited in NCBI's Gene Expression Omnibus [87 (link)] and are accessible through GEO Series accession number GSE89480.