title: Trabscriptome of Heliothis virescens
design: mRNA was isolated from total RNA samples using oligo-d(T)25 magnetic beads (Dynabeads: Invitrogen, CA, USA).  Once dynabead-mRNA binding is chemically reversed, the mRNA will be used as a template for 1st strand cDNA that will be converted to double stranded cDNA.  The resulting double-stranded (ds) overhang fragments are end-repaired by incubation in the presence of T4 DNA polymerase and Klenow polymerase. The polished fragments are phosphorylated by T4 PNK, followed by the addition of a single ‘A’ base to the 3' end of the blunt-ended phosphorylated fragments.  This ‘A’ base prepares the cDNA fragments for ligation to proprietary adapter oligonucleotides (Illumina single-read or paired-read sequences) which have a ‘T’ base at their 3' end.  Ligation products are size-selected by gel electrophoresis and purification (2% low-range agarose with ethidium bromide).  Following 1-2 hours at 80-110V at room temperature, the library range is visualized under brief UV and the 500bp band will be excised with a clean scalpel.  Purified DNA libraries are subjected to a final PCR amplification step (15 cycles).  All amplified libraries are quantitatively and qualitatively assessed by Nanodrop ND-1000 (Thermo Scientific, DE, USA) UV/Vis spectroscopy, DNA bioanalyzer 2100 (Agilent, CA, USA) microfluidics, and realtime quantitative PCR (Life Technology,CA, USA) to determine sequenceable molecules.