RNA-seq of Rhynchophorus ferrugineus midgut
Total RNA was extracted from the larva using TRIzol Reagent (Invitrogen). Extracted RNA was processed using the E.Z.N.A.HP Total RNA Kit (OMEGA RNA, Invitrogen) to eliminate polysaccharides. RNA purity, quality and concentration were determined using Nanodrop, Qubit 2.0 and Agilent 2100 methods. Messenger RNA was extracted from 6 g (50 ng/ul) of total RNA, using oligo (dT) magnetic beads, fragmentation buffer was added to the beads coated with mRNA and mRNA was broken randomly. mRNA was used to synthetize the first cDNA chain and then subjected to a second amplification to obtain double stranded cDNA. Double stranded cDNA was purified using AMPure XP. Finally, the cDNAlibrary was created by PCR enrichment (NEBNext UltraTM RNA Library Prep Kit) using HiSeq2500 high-throughput sequencing.