title: RNAseq_Fly_fem_eGFP_3
organism: Drosophila melanogaster
description: Protocols: Cells were seeded in Nunc T-25 flasks at a concentration of 2.5 million cells in serum free Gibco Sneider 2.5 million cells were treated with 40 Âµg GFP dsRNA (approx 500bp) and harvested after 4 days of treatment. RNA was extracted with ALL prep DNA/RNA Mini Kit QIAGEN according to the manufacturer instructions. Poly-A RNA isolation, RNA fragmentation, and complementary DNA (cDNA) synthesis protocols were performed as in Yoon and Brem (2010). The cDNA was processed for DNA library preparation according to Parkhomchuk et al. (2009). In summary, the protocol includes RNA fragmentation, first-strand synthesis, and second-strand synthesis using deoxyribo- nucleotide triphosphates (dNTPs) and deoxyuridine triphosphates (dUTPs). End repair, A-tailing, and ligation were then performed as well as size selection for fragments of 300-350 bp. The second strand was cleaved by hydrolysis of uracil in the dsDNA. The resulting strand-specific cDNA was amplified before sequencing.