title: RNA-Seq of Varroa destructor
design: 1.5g RNA per sample was used for library construction. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer's recommendations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and fragmented using divalent cations under elevated temperature in NEBNext First-Strand Synthesis Reaction Buffer (5X). First and second-strand cDNA synthesis were synthesized. After adenylation of 3 ends of DNA fragments, NEBNext Adaptors with hairpin loop structure were ligated to prepare for hybridization, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed and purified, library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed using TruSeq PE Cluster Kit v3-cBot-HS (Illumina, USA) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and paired-end reads were generated.