DNA-seq of sexed Drosophila grimshawi, Drosophila silvestris, and Drosophila heteroneura

We aimed to identify the scaffolds of sex chromosome in Hawaiian Drosophila. We extracted genomic DNA and conducted DNA-seq for single sexually-mature fly of Drosophila grimshawi (G1 strain), Drosophila silvestris (Y11R6 strain), and Drosophila heteroneura (W48B6 strain). The DNA extractions were conducted following this pipeline: (1) homogenize one fly in a 1.7 mL microcentrifuge tube (Denville Scientific, Holliston MA) with 250 μL of a solution with 0.1 M Tris HCl (Quality Biological, Gaithersburg MD), 0.1 M EDTA (Quality Biological, Gaithersburg MD), and 1% SDS (Invitrogen, Carlsbad CA); (2) incubate at 70 oC for 30 min; (3) add 35 μL Potassium Acetate (Mallinckrodt, St. Louis MO) and shake; (4) incubate on ice for 30 min; (5) centrifuge at 13,000 rpm for 15 min; (6) transfer supernatant to a new microcentrifuge tube; (7) add 250 μL of Phenol-Chloroform (Sigma, St. Louis MO); (8) centrifuge at 13,000 rpm for 5 min; (9) repeat step 6-8; (10) transfer supernatant to a new microcentrifuge tube; (11) add 150 μL of isopropanol (J.T. Baker Analyzed, Phillipsburg NJ) and shake. (12) centrifuge at 13,000 rpm for 5 min; (13) discard supernatant without touching the pellet; (14) wash the pellet with 1 mL 70% Ethanol (Warner-Graham, Cockeysville MD); (15) centrifuge for 5 min at 13,000 rpm; (16) remove supernatant and let the pellet dry for 20 min; (17) resuspend the pellet with 100 μL nuclease-free water (Quality Biological, Gaithersburg MD). The DNA-seq library were conducted strictly following the Nextra DNA (Illumina, San Diego CA; cat # FC-121-1030) pipeline. We performed single-end 76 bp PolyA+ sequencing on the HiSeq2000 Sequencing System (Illumina, San Diego CA). De-multiplexed reads (Chastity > 0.6) FASTQ output was from Illumina CASAVA (v.1.8.2).