| Design Description | Females reared on standard cornmeal food at room temperature. 50% of females were allowed to freely mate for three days, the rest were maintained as virgins. Adults were aged 4-5 days then flash frozen in liquid nitrogen and stored at −80° C. Total RNA was isolated from whole tissue using the RNeasy Plus Kit (Qiagen, Valencia, CA) per manufacturer’s recommendations. RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality was assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). The TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer’s protocols. Briefly, polyA mRNA was purified from ~100 ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis. End repair and 3' adenylation was performed on the double stranded cDNA. Illumina indexed adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200 to 500 bp in size. The amplified libraries were purified by AMPure (Beckman Coulter, Brea, CA) purification and hybridized to an Illumina pair end flow cell for cluster amplification using the cBot (Illumina, San Diego, CA) at a concentration of 8 picomoles per lane. |
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