SRA

RNAseq was carried out, using Anopheles funestus FANG colony mosquitoes, to compare the effects of poly(A) selection and Ribo-Zero depletion on the observed transcriptomes and to improve the genome annotation. The libraries were generated as follows. Total RNA was extracted from three pools of 10 individual adult female mosquitoes using the Arcturus PicoPure RNA isolation kit (Life Technologies, Carlsbad, USA), according to the manufacturer’s instructions and including a DNase treatment step. On visual inspection of the purified RNA, the samples displayed some pigmentation, the likely effect of which on data quality was unknown. To assess this, the most abundant sample (FANG-1) was split in two. One half of the sample was cleaned using Agencourt AMPure XP beads (Beckman Coulter, Brea, USA) prior to further processing. All four samples were split in two and one of each pair subjected to poly(A) mRNA enrichment and the other to ribosomal RNA depletion. Polyadenylated RNA was selected from total RNA samples using 3 rounds of poly(A) selection with the Dynabeads mRNA purification kit (Life Technologies), using 1.5 µg of starting material. Total RNA was rRNA-depleted using the Ribo-Zero low input kit for Human/Mouse/Rat (Illumina, San Diego, USA), using 100 ng of starting material. RNAseq libraries were prepared from poly(A) and Ribo-Zero mRNA-enriched material using the ScriptSeq v2 RNAseq library preparation kit (Illumina), using 15 cycles of PCR amplification. Libraries were purified using Agencourt AMPure XP beads (Beckman Coulter). Each library was quantified using a Qubit fluorometer (Life Technologies) and the size distribution assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). The eight final libraries were pooled in equimolar amounts using the Qubit and Bioanalyzer data. The quantity and quality of each pool was assessed by Bioanalyzer and subsequently by qPCR using the Kapa Illumina library quantification kit (Kapa Biosystems, Wilmington, USA), on a Light Cycler LC480II (Roche, Basel, Switzerland), according to manufacturers' instructions. The pool of libraries was sequenced on one lane of the HiSeq 2500 (Illumina) at 2x125 bp paired-end sequencing with v4 chemistry.