title: GSM1848649: High Feeder Pop 2, G9 Males, Biological Rep 2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: We flash froze at -80 °C three-to-five day-old virgin flies at generations nine and ten, with at least thirty flies per replicate and two replicates per sex per selected population per generation (32 samples) such that each generation of each population and each sex had two biological replicates that were processed separately. All flies were collected between 1 PM and 3 PM. Total RNA was extracted with Trizol using the RNAeasy Mini Kit (Qiagen, Inc.). rRNA was depleted using the Ribo-Zero™ Gold Kit (Epicentre, Inc.) with 5ug total RNA input. Depleted mRNA was fragmented and converted to first strand cDNA. During the synthesis of second strand cDNA, dUTP instead of dTTP was incorporated to label the second strand cDNA. cDNA from each RNA sample was used to produce barcoded cDNA libraries using NEXTflex™ DNA Barcodes (Bioo Scientific, Inc.) with an Illumina TrueSeq compatible protocol. Library size was selected using Agencourt Ampure XP Beads (Beckman Coulter, Inc.) and centered around 250 bp with the insert size ~130 bp. Second strand DNA was digested with Uracil-DNA Glycosylase before amplification to produce directional cDNA libraries. Libraries were quantified using Qubit dsDNA HS Kits (Life Technologies, Inc.) and Bioanalyzer (Agilent Technologies, Inc.) to calculate molarity. Libraries were then diluted to equal molarity and re-quantified. Two pools of 16 libraries were made, one of the 16 libraries from replicate 1 high and low lines; and one of the 16 libraries from replicate 2 high and low lines. Pooled library samples were quantified again to calculate final molarity and then denatured and diluted to 14pM. Pooled library samples were clustered on Illumina cBot; each pool was sequenced on one lane of Illumina Hiseq2500 using 125 bp single-read v4 chemistry.
attributes.tag: GEO Accession
attributes.value: GSM1848649