title: DNA-Seq of D. melanogaster: C2-F1-75
design: For each sample, 30 female flies of each sx were flash-frozen.  DNA was extracted using a cell lysis solution [1.58 g of Tris-HCl (Quality Biological, Gaithersburg, MD), 37.22 g EDTA disodium salt (Quality Biological, Gaithersburg, MD) and filled to 1 liter with RNase/DNase-free water, adjusting the pH to 8.0 with 10 M NaOH (Sigma Aldrich, St. Louis, MO) when necessary].  Flies were homogenized using an Omni Bead Ruptor (Omni International, Kennesaw, GA).  The solution was incubated with Proteinase K (Life Technologies, Grand Island, NY) at 65C for 1 hour.  The lysate was RNase A treated (20 mg/ml) (Life Technologies, Grand Island, NY) by mixing and incubating at 37C for 15 minutes.  Protein precipitation was with Ammonium Acetate (Quality Biological, Gaithersburg, MD) solution to samples chilled on ice for 5 minutes.  100% isopropanol (VWR International, Radnor, PA) was added and mixed to precipitate the DNA; samples were incubated for 1 hour at -20C.  The DNA pellet was washed with 75% ethanol (NIH Supply Center, Gaithersburg, MD), then re-hydrated in RNase/DNase-free water. DNA samples were then purified using phenol-chloroform extraction. DNA concentration and quality were determined with Nanodrop 8000 (Thermo Fisher Scientific, Asheville, NC). One microgram of genomic DNA were sheared to ~350 bp using a Covaris E210 (Covaris, Woburn, MA) with settings: duty cycle 10%; intensity 5; cycles/burst 200; time 45s. Libraries were constructed using the Tru-Seq DNA PCR-Free LT Sample Prep Kit (Illumina, San Diego, CA) according to manufacturers protocol. The libraries were then pooled and run on a HiSeq2500 (Illumina, San Diego, CA) with version 3 sequencing reagents to generate a minimum of 47 million paired-end 126-base reads per library.  The HiSeq data was processed using RTA1.18.64 and CASAVA 1.8.2.