title: Whole genome sequencing of Drosophila melanogaster: adult whole body
design: We homogenized individual flies in 350 L Lysis Buffer RLT Plus using 2-3 1 mm beads in a bead shaker for 2 minutes. DNA and RNA were extracted using the AllPrep DNA/RNA Micro Kit (Qiagen product number 80284). Following DNA extraction, we purified samples with Ampure XP Beads (Beckman Coulter product number A63880). We prepared 1 L of DNA at ~2.5 ng/L sequencing using a modified Nextera protocol developed by Baym et al. (2015), indexing samples with custom primers, and selecting fragments between 450-500 bp in length using a SizeSelect e-Gel. As a final step to amplify the prepared DNA sequencing libraries, we ran all size selected samples through additional 5 rounds of PCR. Each PCR reaction used 5 L template DNA, 0.6 L of 100 mM forward and reverse primers (custom synthesized by IDT), 10L of KAPA HiFi Ready Mix (KAPA Biosystem product number KK2611/2612), and 3.8 L nuclease free water. Our PCR protocol included 5 minutes of initial denaturation at 95C followed by 4 rounds of 20 seconds denaturation (98C), 20 seconds annealing (62C), 30 seconds elongation (72C), followed by a final elongation at 72C for 2 minutes. Following PCR amplification, we purified DNA libraries using Ampure XP beads and quantified concentrations on a Life Technologies Qubit spectrophotometer and Agilent Bioanalyzer.