cDNA library preparation for transcriptome sequencing

cDNA library were constructed following previous method [17]. Briefly, 3 μg RNA per sample was used as input material for the RNA sample preparation. Sequencing libraries were generated using NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s instructions. Newly isolated mRNA was further purified using with Oligo (dT) magnetic beads and sheared into 200–700 nucleotides sections using fragmentation buffer. The fragmented mRNA was used as templates for first-strand cDNA synthesis using random hexamer primers. Subsequently, second-strand cDNA was synthesized using DNA polymerase I and RNaseH. All remaining overhangs were passivated via polymerase. After adenylation of 3′ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated for hybridization. In order to select cDNA fragments of preferentially 150~200 bp, the library fragments were purified using an AMPure XP system. Then 3 μL USER Enzyme (NEB, USA) was incubated with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by incubation at 95°C for 5 min before PCR reaction. Subsequently, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Amplicons were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The cDNA library of P. interpunctella was sequenced on Illumina Hiseq™ 2500 using paired-end technology in a single run by Beijing Biomake Company (Beijing, China).