title: RNA-Seq of Papaipema moths: speciosissima adult thorax
design: Total RNA was extracted from a single adult moth of each species. Adult moths were collected at UV light and frozen live and without buffer in liquid nitrogen vapor. Samples were later transferred to a -80C freezer, where they were held until RNA extraction. Following dissection on a cryo-cooled work surface, we used half of a thorax for each extraction. RNA was isolated using a standard Trizol protocol (https://tools.thermofisher.com/content/sfs/manuals/trizol_reagent.pdf), then processed on RNAEasy columns (Qiagen) to remove salts and other contaminants. We employed the Agilent RNA 6000 Nano assay on a BioAnalyzer to assess the overall quality of the extracted RNA and generate RIN values, and obtained accurate quantitation with the Qubit RNA BR (broad range) Assay. Total RNA was delivered to the New York Genome Center, where sequencing libraries were prepared using Illumina's TruSeq stranded mRNA kit. Paired-end, 125bp cDNA libraries were sequenced on an Illumina HiSeq 2500, generating ~90 million reads per library.