RNAseq was carried out, using Anopheles funestus (sensu stricto) mosquitoes. Mosquitoes used in the study were insectary-reared F1 progeny of field-caught mosquitoes from four locations in Ghana, Cameroon, Uganda and Malawi. In addition, mosquitoes from the fully insecticide susceptible FANG colony were sequenced as a comparator. In all cases, 3-5 day old female F1 mosquitoes were subjected to WHO bioassays to expose them to permethrin for 1 hour, then maintained in the absence of exposure for a further 24 hours and surviving mosquitoes used for whole transciptome sequencing. Total RNA was extracted from pools of 10 mosquitoes using the Arcturus PicoPure RNA isolation kit (Life Technologies, Carlsbad, USA), according to the manufacturer’s instructions and including a DNase treatment step. 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 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). Libraries were pooled in equimolar amounts (with other libraries not included in this study) and each pool of libraries sequenced on one lane of the HiSeq 2500 (Illumina) at 2x125 bp paired-end sequencing with v4 chemistry.
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