E-MTAB-9465 - RNA-seq of Drosophila simulans and Drosophila mauritiana developing male genitalia
Last updated on 19 August 2020, released on 29 August 2020
Drosophila mauritiana, Drosophila simulans
We generated RNA-seq data of Drosophila simulans and Drosophila mauritiana developing male genitalia in order to identify expression level differences between these species. These species are closely related, yet have dramatic differences in their male genital morphologies. Three independent RNA-seq library replicates were generated for Dsim w501 and Dmau D1 developing male genitalia. Flies were reared under the above conditions, and white pre-pupae collected. Males were selected using gonad size and allowed to develop in a humid container at 25ºC until stages 2 and 4.5 (see staging guide in (Hagen et al., 2019); tartan underlies the evolution of Drosophila male genital morphology). Between these stages, the claspers develop from a ridge structure to a distinct appendage separate from the surrounding tissue, and the posterior lobe has begun to extend outwards from the lateral plate primordia (Hagen et al., 2019). The heads of pupae were impaled with a needle onto a charcoal agar plate and submerged in 1xPBS. Dissection scissors were used to remove the distal tip of the pupal case and the outer membrane, and pressure applied to the abdomen to allow the developing genitalia to be quickly expelled from the pupal case and dissected away from the abdomen. Note that the entire genital arch, including internal genital organs (but not including abdominal tissue), was isolated for RNA extraction. The genitalia from fifteen males from each stage were collected and placed directly into TRIzol. RNA was then extracted using standard procedures. Quality and quantity of RNA was verified using a Qubit, and samples were sent to the Centre for Genomic Research at the University of Liverpool where dual-indexed, strand-specific RNA-seq libraries were prepared using NEBNext polyA selection and Ultra Directional RNA preparation kits. Samples were then sequenced using Illumina HiSeq 4000 (paired-end, 2x150 bp sequencing). Dsim w501 and Dmau D1 reads were mapped against reannotated reference coding sequences (Torres-Oliva et al., 2016).
RNA-seq of coding RNA, development or differentiation design, species design
Unravelling the genetic basis for the rapid diversification of male genitalia between Drosophila species. Joanna F. D. Hagen, Cláudia C. Mendes, Shamma R. Booth, Javier Figueras Jimenez, Kentaro M. Tanaka, Franziska A. Franke, Luis. Baudouin-Gonzalez, Amber M. Ridgway, Saad Arif, Maria D. S. Nunes, Alistair P. McGregor.