D. yakuba reference RNA-seq virgin male carcass, paired end reads
Fly stocks were incubated under controlled conditions at 25and 40% humidity. Virgin flies were collected within 2 hrs. of eclosion, then aged 2-5 days post eclosion before dissection. We dissected samples in isotonic Ringers solution, using female ovaries and headless gonadectomized carcass from two adult flies as well as testes and male headless gonadectomized carcass for four adult flies for each sample RNA prep. We collected three biological replicates of the D. yakuba reference, three biological replicates of the D. simulans w501 reference, and one replicate of the D. ananassae reference (stock numbers in Table 4). Samples were flash frozen in liquid nitrogen immediately after dissection, and and stored in 0.2ml Trizol at -80. All samples were homogenized in 0.5ml Trizol Reagent (Invitrogen) with plastic pestle in 1.5ml tube, mixed with 0.1ml chloroform, and centrifuged 12,000g 15min at 4oC, as Trizol RNA extraction protocol. The RNAs in the supernatant about 0.4ml were then collected and purified with Direct-Zol RNA MiniPrep Kit (Zymo), followed the protocol. The total RNAs were eluted in 65μL RNase-Free H2O. About 1μg purified RNAs were treated with 2μL Turbo DNase (Invitrogen) in 65μL reaction, incubated 15min at room temperature with gentle shaking. These RNAs were further purified with RNA Clean and Concentrator-5 (Zymo). One extra wash with fresh 80% ethanol after the final wash step was added into the original protocol. The treated RNAs were eluted with 15μL RNAse-Free H2O, and stored at -80.  The amplified cDNAs were prepared from 100ng DNase treated RNA with Ovation 4  RNA-Seq System V2 (Nugen) and modified protocol. The preparations followed the protocol to the step of SPIA Amplification (Single Primer Isothermal Amplification). The amplified cDNAs were first purified with Purelink PCR Purification Kit (Invitrogen, HC Binding Buffer) and eluted in 100μL EB (Invitrogen). These cDNAs were purified again to 25muL EB with DNA Clean and Concentrator -5 Kit (Zymo) for Nextera library preparation. About 43ng cDNAs were used to construct libraries with Nextera DNA Sample Preparation Kit (Illumina) and modified protocol. After Tagmentation, Purelink PCR Purification Kit with HC Binding Buffer was used for purification and eluted with 30μL EB or H2O. The products (libraries) of final PCR amplification were purified with DNA Clean and Concentractor-5 and eluted in 20μL EB. The average library lengths about 500bp were estimated from profiles of Bioanalyzer (Agilent) with DNA HS Assay. All libraries were normalized to 2-10nM based on real-time PCR method with Kapa Library Quant Kits (Kapa Biosystems). The qualities and quantities of these RNAs, cDNAs and final libraries were measured from Bioanalyzer with RNA HS or DNA HS Assays and Qubit (Invitrogen) with RNA HS or DNA HS Reagents, respectively. Samples were barcoded and sequenced in 4-plex on an Illumina HiSeq 2500 using standard Illumina barcodes, resulting in high coverage.    Reference:   Rogers, R. L., L. Shao, J. Sanjak, P. Andolfatto and K. R. Thornton (2014) Sex-biased expression in the Drosophila melanogaster group. G3: Genes, Genomes, Genetics. 4: 2345-2351