RNA-seq of Cochliomyia hominivorax larva resistant to OP insecticide
The 3' RNA-seq library was constructed by the Fasteris enterprise using the procedure adapted from a previous study (Torres et al, 2008. Genome Research, 18, 172-177). In this method, 4 μg of total extracted RNA from both samples (control and resistant) were used to create the 3'RNA libraries. A 3'RNA library contains only the RNA fragments possessing the poly A tail. To construct it, the mRNA-Seq Sample Prep Kit (Illumina) was modified to select the 3'RNA fragments. Basically, the mRNA molecules were fragmented at high temperature (80°C) by divalent cations using the fragmentation buffer. The polyA mRNA fragments were purified using poly-T oligonucleotide baits attached to magnetic beads. After the selection of poliA fragments, the mRNA- seq Sample prep Kit (Illumina) protocol was followed according to the manufacturer's instructions. Consequently, we obtained one polyA-fragment per transcript, which allowed us to directly estimate the expression level of the transcripts. Resistant and control samples were pooled prior to sequencing by Fasteris.