Phusion polymerase master mix

First-strand cDNAs were synthesized from 1 μg of total RNA using the SuperScript IV cDNA synthesis kit (Thermo Scientific). The cDNA products were diluted to 50 μL. PCR amplification (50 μL) was performed using 2.5 μL of cDNA as template with attB-flanked gene-specific primers (Additional file 7: Table S5) in a Veriti Thermo Cycler (Thermo Fisher Scientific) using Phusion polymerase master mix (Thermo Fisher Scientific). Cycling parameters were as follows: an initial denaturing step at 94 °C for 5 min, 35 cycles at 96 °C for 15 s, 55 °C for 30 s, 72 °C for 1 min followed by a final extension step at 72 °C for 10 min. Specific PCR products were cloned into the pDONR221 vector (Thermo Fisher Scientific) in the presence of BP clonase (Invitrogen). Plasmid DNAs were isolated according to standard protocols and recombinant plasmids were subjected to sequencing using universal M13 primers and the BigDye terminator cycle sequencing kit v1.1 (Thermo Fisher Scientific). Sequencing products were EDTA/ethanol-precipitated, dissolved in formamide, and loaded for analysis on an in-house capillary 3130xl Genetic analyzer (Applied Biosystems).