454EST from Spodoptera frugiperda fat bodies
Larval fat bodies and midgut were obtained by dissection of eight to ten 3rd - 4th instars individuals from each of five selected insecticide-resistant strains of Spodoptera frugiperda. Total RNA was isolated from midgut (IM) and fat bodies (TA) using the SV Total RNA Isolation System kit (Promega), according to the manufacturer's guidelines, and RNA concentration was determined in a Nanodrop-1000 Spectrophotometer version 3.7.1 (Thermo Scientific, FL). Three µg of total RNA aliquots from each strain were pooled into a single batch sample (15 µg). A 10 µL aliquot of the resulting pool was enriched for mRNA using the RiboMinusTM Eukaryote Kit (Invitrogen) following the guidelines of the manufacturer. The concentration of the enriched mRNA was measured as before, and the obtained RNA stored at -80°C. The RNA obtained was submitted for sequencing on a 454 GS-FLX platform, RNA was further evaluated for quality and integrity, subjected to fragmentation, size selection, library preparation and sequencing using the Titanium chemistry, following the manufacturer guidelines.