Both forewings and hindwings are rapidly dissected and stored in RNA later at −80 ° C (Life Technologies).RNA isolation was performed using Ambion Purelink RNA Mini Kit (Life Technology). RNA-seq libraries were generated according to the manual of NEBNext Ultra RNA library Prep Kit for Illumina (New England Biolabs). Briefly, Poly(A) RNA was isolated with Oligo d(T)25 beads from total RNA, fragmented into 200-400 bp, added into first- and second-strand cDNA synthesis, end repaired, tailed with 3 dA, ligated with an adaptor, barcoded with a unique illumine index, selected with ~200 bp fragments using AMPure XP beads (Beckman, Fullerton, CA), and amplified with 15 cycles of PCR. Libraries were further quantified on a Qubit Fluorometer, pooled, and sequenced on Illumina Nextseq 500 lanes as 36 bp paired-end reads.
GEO Accession
GSM2609367