RNA-seq of Drosophila melanogaster Oregon-R-modENCODE, Eight Day Adults, Whole organism, Pesticide treatments
RNA was isolated using the RNeasy Plus Mini Kit (QIAgen) from 18-30mg of whole frozen flies per sample. The standard RNeasy Plus protocol was used  with the following minor modifications: the lysate was centrifuged for 8 min at 11,750 x g and RNA was eluted with 35 L of buffer. Prepared RNA was quantified using the Bioanalyzer Chip (RNA nano 6000) and the Qubit fluorometer. The smaller of the two concentrations was used to calculate the amount of RNA needed for RNA library preparation (500 ng). The NEBNext Ultra Directional RNA Library Preparation Kit (New England Biolabs) together with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs) was used for RNA library preparation with the following modifications: Section 1.2 mRNA Isolation, Step 37, Fragmentation and Priming Total RNA, we decreased the incubation time from 15 min to 5 min; Section 1.3 First Strand cDNA Synthesis, Step 2, we increased the incubation time from 15 min to 50 min; for the size selection we used an insert size of 300-450bp and a final library size of 400-550bp; and finally in Section 1.9A, PCR library Enrichment, Step 2, we used 14 cycles for PCR cycling. RNA was stored at -80 C prior to RNA sequencing.