Anopheles aquasalis larvae RNAseq
Mosquitoes - An. aquasalis colony (gift from Dr. Paulo Filemon Paolucci Pimenta/Fiocruz) was maintained in the insectary at the Department of Parasitology, ICB-USP (São Paulo, Brazil) at constant temperature, 27±1oC, 75-80% relative humidity and 12 h light:12 h dark photoperiod. Larvae were kept in 0.2% marine salt (w/v), and were fed with powdered fish food (Tetramin, Blacksburg, VA, USA). Adult males and females were kept together in a cage with access to 10% sucrose solution (w/v) ad libitum. Female mosquitoes aged 5-7 days after emerging were allowed to feed on anaesthetized mice for 30 minutes. Eggs collected 2-3 days post blood meal were hatched in 0.2% marine salt (w/v).   RNA extraction and quantification – Larvae RNA was extracted from a pool of third and fourth instar larvae (20 of each). Frozen animals were used for mRNA extraction, using magnetic beads covalently bound to oligo(dT) tags (Dynabeads mRNA DIRECT, Invitrogen, Grand Island, NY, USA), accordingly to the manufacturer’s instructions. Aliquots of the purified mRNA samples were quantified using Quant-iT RiboGreen RNA Reagent (Invitrogen) and their integrity was checked a microfluidics-based platform (Agilent 2100 Bioanalyzer, Santa Clara, CA, USA).     Sequencing – Approximately 400 ng of polyA+ RNA from each sample was used as template for sequencing. mRNA was fragmented with zinc chloride, resulting in molecules with a size distribution range from 300 to 800 bases (assessed by Bioanalyzer), and used as a template for cDNA synthesis. Adaptors were linked to the fragments ends. Beads coated with oligonucleotides that are complementary to the adaptors sequences were incubated with the cDNA fragments, and a water-in-oil emulsion was produced, followed by emulsion PCR. Washed beads were deposited in the picotiter plate wells, and other sequencing reagents were loaded on the 454 GS-Junior sequencer (Roche, Branford, CT, USA). Two hundred sequencing cycles were performed. Base calling was performed by the 454 GS-Junior data processing software GS Run Processor.