Computational protocol: The ß-importin KAP8 (Pse1/Kap121) is required for nuclear import of the cellulase transcriptional regulator XYR1, asexual sporulation and stress resistance in Trichoderma reesei

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Protocol publication

[…] Library preparation and Illumina sequencing were performed at the École Normale Supérieure Genomic Platform (Paris, France). Messenger (polyA+) RNAs were purified from 800 ng of total RNA using oligo(dT). Libraries were prepared using the stranded RNA-Seq library preparation TruSeq RNA Sample Prep Kits (Illumina). Libraries were multiplexed by four on three single flow cell lanes and subjected to 50 bp paired-end read sequencing on a HiSeq 2000 device. A mean of 47 ± 6 million passing illumina quality filter reads was obtained for each of the 12 samples. Transcriptomic analysis was performed as three experimental replicates from which mean values were calculated and presented. RNA-Seq data analysis was done using the Eoulsan software version 1.2.2 (Jourdren et al., ). Before mapping, poly N read tails were trimmed, reads ≤ 40 bases were removed, and reads with quality mean ≤ 30 were discarded. Reads were then aligned against the T. reesei reference genome (http://genome.jgi-psf.org/Trire2/Trire2.home.html) using Bowtie mapper (version 0.12.7) using the ‘-n 2 -l 34 -e 70 -k 2 --best’ parameters. Only one alignment was kept in a given locus for each read, and read alignments matching on more than one locus were removed. To compute gene expression, the T. reesei genome annotation was used Gene expression was computed by counting all overlapping regions between alignments and referenced exons. To quantify the gene expression level, the relative transcript abundance was measured in reads per kilobase of exon per million mapped sequence reads (RPKM; (Mortazavi et al., )). The log2 ratios of the RPKM values were used to identify differentially expressed genes. To keep only the most differentially expressed genes, thresholds of four (sophorose vs. glycerol in the retransformant) and five (retransformant vs. Δkap8 on sophorose) for log ratio were used. Read numbers < 100 were considered as (almost) absence of transcription and not chosen for the evaluation. Genes were identified by the aid of a completely manually annotated T. reesei genome database proprietary to C.P.K.The RNA-Seq gene expression data and raw fastq files are available at the GEO repository (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE59600. […]

Pipeline specifications

Software tools Eoulsan, Bowtie
Application RNA-seq analysis
Organisms Trichoderma reesei, Aspergillus nidulans, Saccharomyces cerevisiae