title: RNA-Seq: head and salivary glands
design: Total RNA was extracted from salivary heads and glands salivary tissues of 5th instar male nymphs of M. garjadoi (n = 6), M. spinolai (n = 6), and M. parapatrica (n = 6) (Table 1). The heads and glands salivary of the male nymphs were carefully removed to preserve the integrity of the salivary glands. Extraction was carried out according to the modified Trizol/Chloroform protocol (Chomczynski and Mackey, 1995). RNA integrity was checked visually on an agarose gel and quantified using a Qbit fluorometer, while NanoDrop measurements ensured the selection of samples with indices close to 2.0 at the 260/230 and 260/280 ratios. In addition to checking integrity by RNA integrity number - RIN, obtained by capillary electrophoresis using Agilent 2100 Bioanalyzer System, (Agilent Tecnologies, Standard Protocol for RNA). RNA-Seq libraries were prepared from samples of the species studied using the TruSeq RNA Sample Prep Kit v2 (Ilumina) protocol according to the manufacturer's instructions. Libraries (n =18) were grouped into 12 and run in one lane of a flow cell on the Illumina HiSeq2500 platform with 2x100 bp paired-end (Laboratrio de Genmica Funcional Aplicada  Agricultura e Agroenergia, ESALQ-USP, Brazil).