nymph_wing
The wing discs of L. migratoria fifth instar nymph were dissected along the wing root with small scissors under microscope. Thirty pairs of dissected wing discs were combined, and total RNA samples were prepared using TRizol Reagent (TIANGEN, Beijing, China) following the manufacturer’s instructions. Ten ug of total RNA was used to isolate mRNA using oligo(dT) magnetic beads. The cDNA library was constructed using NEBNext® mRNA Library Prep Reagent Set (NEB, Ipswich, MA, USA) following the manufacturer’s protocols. Briefly, enriched poly(A) RNA of each sample was fragmented into 200-700 nt pieces with RNA Fragmentation Reagents. The cleaved RNA fragments were transcribed into the first-strand cDNA using random hexamer-primers, followed by second-strand cDNA synthesis. The resulting double-stranded cDNA (dsDNA) was purified with QiaQuick PCR extraction kit (Qiagen, Hilden, Germany) and resolved in EB buffer. The purified dsDNA was treated with T4 DNA Polymerase and T4 Polynucleotide Kinase for end-repairing and dA-tailing. After that, they were ligated to sequencing adaptors with barcode using T4 DNA ligase. Finally, fragments with around 200bp-length were purified with QiaQuick GelPurify Kit (Qiagen, Hilden, Germany), and used as templates for PCR amplification to create the cDNA library. The library was sequenced on Illumina HiSeq™ 2000 (Illumina, San Diego, CA, USA)