Computational protocol: RNA Sequencing Reveals Xyr1 as a Transcription Factor Regulating Gene Expression beyond Carbohydrate Metabolism

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Protocol publication

[…] Fungal mycelia of four samples were harvested by filtration and centrifugation, frozen, and ground under liquid nitrogen. Total RNA were isolated by TRIzol (Invitrogen) according to the instruction, and RNA extracts were monitored by electrophoresis and quantified using a spectrophotometer.Total RNA were provided to Chinese National Human Genome Center at Shanghai for sequencing. mRNA purified from total RNA using the MicroPoly(A)Purist kit (Ambion) was used for library preparation and latter sequencing using the Illumina Hiseq2000 platform. The quality control was performed with FASTX-Toolkit (Version 0.0.13), and the reads were filtrated when more than 20% of bases with PHRED score were lower than 30. The cleaned RNA-seq reads were mapped to the T. reesei Rut-C30 genome using Tophat V1.3.2 [] with Bowtie V0.12.7.0 [] with a minimum intron size of 20 bp and a maximum intron size of 100,000 bp; the mean inner distance between mate pairs and the standard deviation for the distribution on inner distances between mate pairs were set to 150 and 75, respectively. Transcript abundance indicated as the number of fragments per kilobase per million fragments mapped (FPKM) was estimated with the program Cuffdiff from the package Cufflinks v0.83 []. Differential expression values were determined using DEseq [, ]. FDR (False Discovery Rate) control (the cutoff was 0.01) was used to identify the significance of differences for multiple tests []. The genome sequence and gene structure information for T. reesei QM6a (http://genome.jgi.doe.gov/Trire2/Trire2.home.html) and Rut-C30 (http://genome.jgi.doe.gov/TrireRUTC30_1/TrireRUTC30_1.home.html) were obtained from the JGI genome Portal site [, ]. The FunCat [] annotation for QM6a was downloaded from the PENDANT genome database []. The FunCat annotation for Rut-C30 was performed using a blastn [] search between QM6a and Rut-C30. The differentially expressed genes were further fine-sorted after being classified according to their FunCat annotations. […]

Pipeline specifications

Software tools FASTX-Toolkit, TopHat, Bowtie, Cufflinks, DESeq, BLASTN
Databases FunCat JGI Genome Portal
Application RNA-seq analysis
Organisms Trichoderma reesei
Diseases Epilepsy, Tonic-Clonic
Chemicals Adenosine Triphosphate, Amino Acids, Glucose, Lactose