Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance
Total RNA was isolated with Trizol (Invitrogen) from whole bodies of 20 larvae, 10 adult females and 10 adult males. Extracted RNA was treated with TerminatorTM 5'-Phosphate-Dependent Exonuclease to produce mRNA-enriched samples by selectively digesting the ribossomal RNA. Genomic DNA contamination was removed by DNase I and the mRNA-enriched samples were further purified by using Nucleospin RNA Clean-up columns. First-strand cDNA was generated from approximately 0.3 µg of mRNA using the SMART approach. The resulting double-stranded cDNAs were normalized using a duplex-specific nuclease enzyme. MID larvae: CGTGTCTCTA; MID adult females: TAGTATCAGC; MID adult males: CTCGCGTGTC.