RNA-Seq of Sphenophorus levis: larvae
Midgut RNA from 30-day-old larvae was treated with RNAse-free DNAse I (Invitrogen) in reactions with 3 g of RNA to a final volume of 20 L. poly (A) mRNA was isolated with oligo(dT) beads and transcriptome construction was carried out with TruSeq Small RNA Library Preaparation Kits (Illumina), according to the manufacturers instructions. Briefly, mRNA was fragmented in Fragmentation Buffer 1x (Illumina) and used as template in a reaction with SuperScript II (Invitrogen) using random hexamer-primers to obtain the first-strand cDNA, followed by a reaction with DNA polymerase I and RNAse H (Illumina) to synthesize the double-strand cDNA. After pair-ending, adenylation and ligation, suitable fragments (200  30 bp) were selected and used for constructing libraries further sequenced using HiSeq1000 (Illumina) in pairedend reads (2x100 bp).