Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNA was isolated from first instar larvae, fifth instar larvae and adults using the TRIzol (Invitrogen) reagent by following the company manual. Poly(A) mRNA was isolated from 20 µl total RNA using Oligo (dT) magnetic beads and then was broken into short fragments (about 200bp) in the presence of fragmentation buffer at 94 °C for 5 min. The short fragments (about 200 bp) were used as templates for first-strand cDNA synthesis using random hexamer-primers. Subsequently, second-strand cDNAs were synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. After purification of short fragments with a QiaQuick PCR Purification Kit (Qiagen), samples werethen washed with EB buffer for end reparation and single nucleotide adenine addition. Finally, the short fragments were connected to sequencing adapters. Suitable fragments (about 200bp), as judged by agarose gel electrophoresis, were enriched with PCR amplification to prepare the sequencing library.