A mixture of eight DCV isolates was passaged three times in Wolbachia-infected or Wolbachia-free Drosophila melanogaster females.

Total RNA was extracted from flies at the end of the selection experiment (Passage 3). Since the DCV genome contains a poly(A) tail, we isolated the virus genomic RNA along with fly transcripts by capturing the polyadenylated RNAs from the extracted total RNA using the KAPA Stranded mRNA-Seq kit (Kapa Biosystems) and a different indexed adapter for each RNA library. Twenty four libraries were prepared (one per virus population), quantified by qPCR and pooled in equal proportions into a multiplexed library. The pool was sequenced on one lane of Illumina HiSeq4000 to generate single-end 50bp reads.