Responses to different oils in Chrysomya megacephala (Fabricius) using digital gene expression (DGE)
mRNA was purified from 6 µg total RNA using Oligo (dT) magnetic beads, and then double-strand cDNAs were directly synthesized on the magnetic beads using the primer of Oligo (dT). The bead-bound cDNAs were subsequently digested with restriction enzyme NlaIII, which recognized and cut off the CATG sites. The fragments apart from the 3' cDNA fragments connected to Oligo (dT) beads were washed away and the Illumina adaptor 1 was ligated to the sticky 5' end of the digested bead-bound cDNA fragments. The junction of Illumina adaptor 1 and CATG site was the recognition site of MmeI, which cut at 17bp downstream of the CATG site, producing tags with adaptor 1. After removing 3' fragments with magnetic beads precipitation, Illumina adaptor 2 was ligated to the 3' ends of tags, acquiring tags with different adaptors of both ends to form a tag library. The library was amplified by linear PCR for 15 cycles, 105bp fragments were purified by 6% TBE PAGE Gel electrophoresis. After denaturation, the single-chain molecules were attached to the Illumina Sequencing Chip (flow cell) for sequencing. Each tunnel generated millions of raw reads with a sequencing length of 49bp.