alias: AJZ0550
title: DNA-Seq of D. melanogaster: SAIP_G123.11_M
design: For each SAIP culture bottle, 30 females were flash-frozen as one sample; 30 males were flash-frozen as a replicate sample. 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). PCR-free libraries were generated from 1 microgram genomic DNA using a Covaris LE220-plus to shear the DNA and the TruSeq DNA PCR-Free HT Sample Preparation Kit (Illumina) for library generation. The median insert sizes were approximately 400 bp. Libraries were tagged with unique dual index DNA barcodes to allow pooling of libraries and minimize the impact of barcode hopping. Libraries were pooled for sequencing on the NovaSeq 6000 (Illumina) to obtain at least 44 million 151-base read pairs per individual library. The data was processed using RTA version 3.4.4.
identifiers.namespace: SUB7929094
identifiers.id: AJZ0550