3 protocols
normalization data transformation protocol
RNA-seq reads from SOLiD 4 from each of the experimental samples were mapped to the T. reesei genome (JGI T. reesei version 2.0 unmasked assembled scaffolds) using the BioScope 1.3.1 Transcriptome Pipeline (LifeTechnologies). Bioscope mapped each read against the complete genome sequence and exon spanning junctions using available gene coordinate information, hence providing the primary read alignment position. Mapping results were recorded in a BAM (binary alignment/map) format. The amount of reads per gene for each sample was calculated with Htseq-count (http://www.huber.embl.de/users/anders/HTSeq) using the BAM files and the genome annotation. Strand-specific RNA-seq reads, as generated by SOLiD, can be specified for when executing Htseq-count in determining accurate read counts per gene. Gene counts were used to calculate normalised expression values (reads per kilobase of exon model per million mapped reads = RPKM) for each gene (Mortazavi et al., 2008). Antisense transcription was detected by either excluding or including strand-specificity in the calculations when comparing the Htseq-count generated counts. Genome_build: JGI T. reesei version 2.0 genes filtered models Supplementary_files_format_and_content: Tab-delimited text files include Htseq-counts and RPKM values for each sample.
nucleic acid library construction protocol
Mycelia from each condition were snap-frozen and ground to a fine powder under liquid N2 using a mortar and pestle. The TRIzol reagent protocol (Invitrogen) was used to extract total RNA from mycelial powder samples. RNA was further purified with the Qiagen RNeasy® Mini Kit (Qiagen) following the instructions of the “RNA clean-up” protocol with on-column DNA digestion. Ribosomal RNA was degraded in 10 µg of total RNA using the Ribominus Eukaryotic kit (Invitrogen, Cat. No. A10837-08). SOLiD whole transcriptome libraries were made according to the SOLiD Whole transcriptome kit protocol (Applied Biosystems, Cat. No. 4425680) and the library concentrations were measured with the Quant-ut HS dsDNA assay kit (Invitrogen, Cat No. Q32851). Libraries were pooled to equimolar amounts (Invitrogen, Cat. No. Q32851) and gel purified using 2% size-select E-gels to 200-300 bp (Invitrogen). Emulsion PCR (0.5 M final concentration of pooled libraries) and bead-based enrichment was done according to the SOLiD 3+ template bead preparation guide containing library. Sequencing was carried out on a SOLiD 3+ ABi sequencer platform according to manufacturer’s instructions to generate 50 bp reads in colour space.
growth protocol
Conidia of T. reesei QM6a were inoculated on potato dextrose agar slopes (PDA: 4.0 g/L potato extract, 15.0 g/L agar, 20.0 g/L) at 28°C until they had grown and sporulated. Spores were resuspended in 2 ml 0.01% (w/v) Tween 80 and and used to inoculate liquid batch cultures at a concentration of 10^5 spores/mL. Strains were cultured in 100 ml Trichoderma Minimal Media [TMM: 15 g/L KH2PO4, 5 g/L (NH4)2SO4, 10 g/L of the respective carbon source, 0.005 g/L FeSO4.7H20, 0.0016 g/L MnSO4.H20, 0.0014 g/L Zn.SO4.H20, 0.0037 g/L CoCl12.6H20, 0.6 g/L MgSO4, 0.6 g CaCl2] in 250 mL Erlenmeyer flasks at 28°C, shaking at 150 rpm. Mycelia were grown for 48 h in 1% (w/v) glucose, after which they were removed by filtration through Miracloth (Merck), washed with double-distilled water (ddH2O), and transferred to fresh media supplemented with 1% w/v of wheat straw for 24 h. Glucose was then added to the wheat straw-incubated cultures to a final concentration of 1% (w/v) and incubation continued for another 5 h.