RNA-seq of salivary glands from Rhipicephalus sanguineus infected with Ehrlichia canis: Sap2
Salivary glands (SG) were dissected from engorged female ticks collected from the infected and uninfected dogs and stored in RNAlater (Ambion) at -20ºC. Total RNA from the SG was extracted using the All-in-One Purification Kit (Norgen Biotek Corporation), following the manufacturer’s instructions. RNA concentration and integrity were evaluated by analysis of rRNA integrity using Agilent 2100 Bioanalyzer (Agilent Technologies Inc.). Libraries preparation was performed with "NEBNext Ultra Directional RNA Library Prep" kit (New England Biolabs) following manufacturer’s instructions. Briefly, magnetic beads with Oligo(dT) were used to enrich poly (A) mRNA from 1 µg of total RNA. Next, the purified mRNA was disrupted into short fragments, and double-stranded cDNA was immediately synthesised. cDNA was subjected to end-repair and adenylation, then connected with sequencing adapters. Suitable fragments, purified by size selection protocol with AMPure XP beads (Beckman Coulter), were selected as templates for PCR amplification. The final library sizes and qualities were evaluated electrophoretically using an Agilent High Sensitivity DNA kit (Agilent Technologies); the fragment size range was 300–1300 bp. Subsequently, the library was sequenced using a HiSeq 2500 sequencer (Illumina) in rapid run mode. Cluster generation was performed, followed by 2x100 cycle sequencing reads separated by a paired-end turnaround. Image analysis was performed using the HiSeq control software version 1.8.4.