The Genetic Basis of Rapidly Evolving Male Genital Morphology in Drosophila

Deep sequencing of total RNA extracted from the genital discs of males for each of the following strains : Drosophila sechellia, Drosophila mauritiana, hybrid introgression line 3Q1(A) and hybrid introgression line Q1(A) Overall design: Analysis of poly(A)+ RNA for three independent biological replicates of sequencing libraries for each of the following strains: D. sechellia w, D. mauritiana P-insertion Q1, hybrid introgression line 3Q1(A), and hybrid introgression line Q1(A). Male genital discs were obtained as described above, and total RNA was extracted using RNAqueous®-Micro Kit (Ambion). Poly(A)+ transcripts were isolated subsequently using MicroPoly(A)Purist� Kit (Ambion). To facilitate normalization of reads across our samples, at this stage of library construction we spiked-in small amounts of exogenous RNA from ArrayControl� Kit (Ambion) into each sample of poly(A)+ RNA. Paired-end sequencing was carried out by loading the samples onto four lanes (three samples per lane) of a flow cell and run on an Illumina Genome Analyzer IIx sequencer using 72 cycles per end of each paired-end read. Biological replicates of each genotype were loaded onto separate lanes.