Mastercycler ep realplex 2.2 system

Quantitative PCRs were performed in a Mastercycler^® ep realplex 2.2 system (Eppendorf, Hamburg, Germany). All reactions were performed in triplicate. The amplification mixture (final volume 25 μL) contained 12.5 μL 2×  iQ SYBR Green Mix (Bio-Rad Laboratories, Hercules, USA), 100 nM forward and reverse primer, and 2.5 μL cDNA (diluted 1:100). Primer sequences are provided in Additional file 3. Cycling conditions and control reactions were performed as described previously [31 (link)]. Calculations using sar1 and act1 as reference genes were performed as published previously [31 (link)].

Approximately 20 mg of harvested mycelia were homogenized in 1 ml of peqGOLD TriFast DNA/RNA/protein purification system reagent (PEQLAB Biotechnologie, Erlangen, Germany) using a FastPrep FP120 BIO101 ThermoSavant cell disrupter (Qbiogene, Carlsbad, CA, United States). RNA was isolated according to the manufacturer’s instructions, and the concentration was measured using the NanoDrop 1000 (Thermo Scientific). Synthesis of cDNA from mRNA was carried out using the RevertAid^TM H Minus First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions.
Reverse transcription polymerase chain reactions (RT-PCRs) were performed in a Mastercycler^® ep realplex 2.2 system (Eppendorf, Hamburg, Germany). All reactions were performed in triplicates. The amplification mixture (final volume 25 μl) contained 12.5 μl 2 × iQ SYBR Green Mix (Bio-Rad), 100 nM forward and reverse primer, and 2.5 μl cDNA (diluted 1:100) as template. All used primers are listed in Supplementary Table S1. Cycling conditions and control reactions were performed as described earlier (Steiger et al., 2010 (link)).

Approximately 20 mg of harvested mycelia was homogenized in 1 ml of peqGOLD TriFast DNA/RNA/protein purification system reagent (Peqlab Biotechnologie, Erlangen, Germany) using a FastPrep FP120 BIO101 ThermoSavant cell disrupter (Qbiogene, Carlsbad, CA). RNA was isolated according to the manufacturer's instructions, and the concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Scientific). Synthesis of cDNA from mRNA was carried out using the RevertAid H Minus first-strand cDNA synthesis kit (Thermo Scientific) according to the manufacturer's instructions.
Quantitative PCRs were performed in a Mastercycler ep realplex 2.2 system (Eppendorf, Hamburg, Germany). All reactions were performed in triplicates. The amplification mixture (final volume, 25 μl) contained 12.5 μl of 2× iQ SYBR green mix (Bio-Rad), 100 nM concentrations of the forward and reverse primers, and 2.5 μl of cDNA (diluted 1:100) as the template. All of the primers used are listed in Table 1. Cycling conditions and control reactions were as described previously (23 (link)). Calculations using sar1 and act1 as reference genes were performed as published previously (23 (link)).