title: RNA-Seq of D. melanogaster: adult ovary/testis
design: Briefly, Trizol- homogenized tissue samples were thawed at room temperature and treated with 0.2 volumes chloroform to promote phase separation. RNA was extracted from the aqueous phase using Qiagen RNeasy Plus Mini Kit. This included application of aqueous phase to Qiagen gDNA Eliminator spin columns to limit carryover of genomic DNA. DNA contamination was also addressed by on-column DNAse digestion (Promega RQ1 DNAse). RNA quality and concentration was validated via Agilent Bioanalyzer; RNA quality for all samples was confirmed to have an RQN >=7.0 and at least 1.0 _g of starting material. Stranded cDNA library preparation was performed by Polar Genomics (Ithaca, NY). mRNA was isolated and fragmented from total RNA pools, followed by 1st and 2nd (dUTP incorporated) strand synthesis. dsDNA was subsequently dA-tailed and adaptor-ligated, followed by size selection, UDG digestion to eliminate the second strand, and PCR amplification. Sequenced on a single lane of Illumina HiSeq 2500 High Output (single-end, 50 bp).