RNA Interference Knockdown Protocol

Glutathione S-transferase (GST), cytochrome c oxidase subunit III (COXIII), dynein (DYN), synaptobrevin (SYN) and phosphatidylinositol-3,4,5-triphosphate 3-phosphatase (PHOS) were selected for knockdown studies using RNA interference technique. Primer sets for each gene were designed (Table S1) using Primer3 software [32] (link), [33] (link). Tick guts and salivary glands from adult males were used as a template for generating target gene amplicons. Amplicons were cloned into the TOPO TA Cloning Kit Dual Promoter (with pCRII-TOPO) (Invitrogen). The MEGAscript Transcription Kit was used for dsRNA synthesis following the manufacturer's protocol (Ambion, Austin, TX, USA). The dsRNA molecules were purified, quantified by spectrophotometry, analyzed by gel electrophoresis, and stored at −20°C.