title: DNA-Seq of D. melanogaster: SIP_S2_4
design: For each sample, 30 flies of each sex 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 65°C for 1 hour.  The lysate was RNase A treated (20 mg/ml) (Life Technologies, Grand Island, NY) by mixing and incubating at 37°C 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 -20°C.  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). With the exception of one sample, two micrograms of genomic DNA were sheared to ~550 bp using a Covaris E220 (Covaris, Woburn, MA) with settings: duty cycle 10%; intensity 175; cycles/burst 200; time 80s. Only one microgram of DNA was available for line SIP_L2_2, so the DNA was sheared to ~350 bp using a Covaris E220 with settings: duty cycle 10%; intensity 3; cycles/burst 200; time 60s.  Libraries were constructed using the Tru-Seq DNA PCR-Free LT Sample Prep Kit (Illumina, San Diego, CA) according to manufacturer's 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 10 million paired-end 251-base reads per library.  The HiSeq data was processed using RTA1.18.64 and CASAVA 1.8.2.