title: GSM2187062: C4_151020_run0437_female_HPS_2; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Heads were dissected in RNAlater (Qiagen, Valencia, CA. Cat. No. 1018087), pooled and stored at -20ºC until RNA extractions. Total RNA was extracted using Direct-zol RNA MiniPrep (Zymo, Irvine, CA. Cat. No. R2050) following manufacturers protocol. Libraries were prepared using the Truseq PolyA+ stranded mRNA kit (Illumina, San Diego, CA. Cat. No. RS-122-2101), following the manufacturer’s protocol. We used a half reaction size with 100ng total RNA as input for all samples. External RNA Control Consortium (ERCC) spike-ins transcribed from a certified plasmid collection (SRM2374) were added to the Fragment-Prime-Finish Mix during the mRNA fragmentation step.  We used the previously described RNAs from Pool 78A for DMSO treated flies and 78B for control flies (20pg /sample)  (Zook et al., 2012. PMID: 22859977).  Libraries were multiplexed using 12 differently barcoded adapters from TruSeq (Illumina, San Diego, CA). Libraries were quantified with Quant-iT PicoGreen (Life Technologies, Carlsbad, CA), and pooled for multiplexed sequencing. We sequenced in an Illumina HiSeq2500, to produce single-end, 75 bp reads.  (Illumina, San Diego, CA.).