RNA-seq of Cylas brunneus: second instar larvae
Total RNA was extracted from second instar larvae of Cylas brunneus using the RNeasy Mini Kit (Qiagen). Total RNA was purified using oligo (dT) magnetic beads to isolate poly-A containing mRNA and fragmented into short sequences using divalent cations. The cleaved RNA fragments were then used for synthesis of first and second strand cDNA. After the end repair on the double-stranded cDNA, 3 ends were adenylated and adapters with indexes were ligated for multiplexing. The cDNA library was amplified by PCR, and then AmpureXP beads were used for purification.  The final library was quantified using Agilents Bioanalyzer High Sensitivity DNA Chip prior to clustering on the Illumina cBot. The cDNA libraries were sequenced on the Illumina sequencing platform (HiSeq2000) where each sample was collocated in one lane of a 100bp single-end run.