Qiazol lysis reagent kit

After incubation, the whole fungal colony of A. flavus was removed from the membrane of each float culture and then immediately flash frozen in liquid nitrogen. Total RNA was then extracted from the mycelia using a QIAzol Lysis reagent kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The total RNA was then purified using a Riboclear plus kit (GeneAll, Korea). Quality and quantity of RNA samples were analysed using the Caliper LabChip GX system (Caliper Life Sciences, Hopkinton, MA, USA). RNA samples with quality scores more than 7 were used for transcriptome library construction and RNA sequencing.

Total RNA isolation was carried out using a QIAzol lysis reagent kit (Qiagen) following the manufacturer's procedure. The cDNA was produced with 500 ng total RNA using a cDNA synthesis kit (PrimeScript RT Reagent Kit; TAKARA, Japan). Primers were designed using Allele ID software (PREMIER Biosoft USA, version 7.5;) (See supplementary Table S1). The Real-time PCR was performed in a 10 mL total volume using 5 mL of RealQ Plus 2 × Master Mix Green (Amplicon, Denmark), 0.5 mL of each sense and antisense primer (10 pmol/reaction), and 25 ng cDNA. The qPCR was conducted on a Rotor-gene 6000 PCR system (Corbett Life Science) with the following program: initial denaturation at 95 °C for 15 min, followed by 40 cycles of denaturation at 95 °C for 15 s, and annealing and extension at 60 °C for 45 s. The program was followed by a melt step at 55 °C–94 °C. The relative gene expression was evaluated using the 2^-∆∆CT method, with the eEF1A1 gene used as the internal control.