RNA extraction and RNA-seq library preparation

Total RNA was extracted from mycelial samples using Trizol® (Diamed, Mississauga, Canada) following the manufacturer protocols. Briefly, 50 mg of hyphae per sample were ground to a fine powder in liquid nitrogen and 1 mL of Trizol added to each sample. Total RNA in the supernatant was purified using a chloroform wash, precipitated using isopropanol and dissolved in RNase-free water. Total RNA quantity and quality were measured using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and denaturing formaldehyde gel electrophoresis, respectively. RNA-seq libraries were prepared for each replicate from 4 μg total RNA using the KAPA stranded mRNA-Seq kit (KAPA Biosystems, Inc., Wilmington, MA) with slight modifications. Briefly, mRNA was captured and purified by performing two purifications on the KAPA mRNA capture beads by mixing total RNA with 50 μL of beads, heating at 65°C for 5 minutes and cooling with shaking at 150 rpm at room temperature for 20 minutes. cDNA libraries with fragment lengths of 200–300 bp mRNA were constructed following the manufacturer protocols. The 12 libraries were barcoded using NEXTflex RNA-seq barcodes (BiooScientific, Toronto, Canada) 1 through 12 (v1-1-15-1), and amplified using the following conditions: initial denaturation at 98°C for 45 s, followed by 12 cycles of denaturation at 98°C for 15 s, annealing at 60°C for 30 s and elongation at 72°C for 30 s, and a final extension at 72°C for 5 minutes. Libraries were purified using NucleoMag NGS Clean-up and Size Selection beads (Machery-Nagel, Bethlehem, PA, USA) and quantified by a NanoDrop ND-1000 Spectrophotometer, and the size and quality were confirmed by agarose gel electrophoresis (2%). Libraries were pooled such that one final library contained equal amounts of each of the 12 barcoded libraries and the final quality and quantity were confirmed using a bioanalyzer at the Genome Quebec Research Centre (Montreal, Canada). The library was sequenced at the Genome Quebec Research Centre (Montreal, Canada) and sequenced in one lane using the Illumina HiSeq 2000 system with 100 bp paired-end sequencing.