title: Application of metagenomics and pyrosequencing to facilitate discovery of potential viral natural enemies of the invasive Caribbean crazy ant, Nylanderia pubens (Forel)
design: Total RNA was extracted from samples of colonies of N. pubens by the Trizol (Invitrogen) method according to the manufacturer’s instructions.  Samples were taken from 9 colonies.  A total of 609 ants of different life stages (workers, alates, queens, larvae, pupae, and eggs) were used to prepare the total RNA.  RNA quality of each preparation was assessed by microfluidic analysis on an Agilent 2100 Bioanalyzer (Agilent, Cary, NC) using the RNA 6000 Nano kit according to the manufacturer’s directions.  Microfluidic assays were completed immediately after RNA extraction using a 1 µl volume of purified sample. RNA samples of acceptable quality were pooled and used as source material for mRNA purification.  mRNA was isolated from the total RNA sample using the Oligotex mRNA Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The isolated mRNA was then utilized to prepare a non-normalized fragment library suitable for 454 platform sequencing using the NEBNext mRNA Sample Pre Reagent Set 2 (New England BioLabs, Ipswich, MA) following the manufacturer’s protocol.