Male Sabethes cyaneus salivary glands
Tissue samples were submitted to the North Carolina State Genomic Sciences Laboratory (Raleigh, NC, USA) for Illumina RNA library construction and sequencing. Prior to library construction, RNA integrity, purity, and concentration were assessed using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Chip (Agilent Technologies, USA). Purification of messenger RNA (mRNA) was performed using the oligo-dT beads provided in the NEBNExt Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, USA). Complementary DNA (cDNA) libraries for Illumina sequencing were constructed using the NEBNext Ultra Directional RNA Library Prep Kit (NEB) and NEBNext Mulitplex Oligos for Illumina (NEB) using the manufacturer-specified protocol. Briefly, the mRNA was chemically fragmented and primed with random oligos for first strand cDNA synthesis. Second strand cDNA synthesis was then carried out with dUTPs to preserve strand orientation information. The double-stranded cDNA was then purified, end repaired and a-tailed for adaptor ligation. Following ligation, the samples were selected a final library size (adapters included) of 400550 bp using sequential AMPure XP bead isolation (Beckman Coulter, USA). Library enrichment was performed and specific indexes for each sample were added during the protocol-specified PCR amplification. The amplified library fragments were purified and checked for quality and final concentration using an Agilent 2100 Bioanalyzer with a High Sensitivity DNA chip (Agilent Technologies, USA). The final quantified libraries were pooled for sequencing on one lane of an Illumina HiSeq 2500 DNA sequencer, utilizing 125 bp single end sequencing flow cell with a HiSeq Reagent Kit v4 (Illumina, USA).