GSM2978239: w-25-3d-II; Drosophila melanogaster; RNA-Seq
Flies were anesthetized using CO2, and then decapitated, followed by brain dissection. The brains of >21 male flies were dissected, carefully removing attached eye and trachael tissue in cold PBS. The PBS was then removed, and the remaining brains were ground in Trizol (Thermo Fisher Scientific, Waltham, MA) using a pellet pestle and frozen at -80 degrees C. Total RNA was purified from dissected brains using the Trizol protocol according to the manufacturer. Total RNA was cleaned of residual DNA using DNA-free (Invitrogen, Carlsbad, CA).  DNA-free reactions were then further purified using Rneasy columns (Qiagen, Germantown, MD) and RNA was eluted in DEPC-treated water. Purified total RNA was evaluated on a Bioanalyzer (Agilent, Santa Clara, CA). Total RNA was quantified using Qubit (Thermo Fisher Scientific, Waltham, MA). Approximately 95-200 ng of total RNA was used for each library. RNA libraries were prepared for sequencing using TruSeq mRNA High Throughput Library Prep Kit according to the kit manual (Illumina, San Diego, CA).  Individual libraries were quantified with KAPA Library quant kit and pooled accordingly (KAPA Biosystems, Boston, MA). Before loading, the pooled library was quantified with Qubit (Thermo Fisher Scientific, Waltham, MA) and diluted accordingly. Libraries were sequenced in two runs of a Nextseq 500. Data from the two runs are provided separately as "a" files and "b" files.