title: Pyrosequencing-based expression profiling and identification of differentially regulated genes from Manduca sexta, a lepidopteran model insect with unknown genome sequence
design: Total RNA samples were extracted from induced hemocytes (IH) and fat body (IF) 24 h later using TRIZOL Reagent (Life Technologies Inc.). Control hemocyte (CH) and fat body (CF) RNA was prepared from day 3, 5th instar naïve larvae (60). PolyA+ RNA was separately purified from the total RNA samples (1.0 mg each) by binding to oligo(dT) cellulose twice in the Poly(A) PuristTM Kit (Ambion). First strand cDNA was synthesized using mRNA (5.0 µg), random dodecanucleotides (100 pmol), and SuperScriptTM III reverse transcriptase (1000 U, Life Technologies Inc.). RNase H treatment, second strand synthesis, and gap joining were performed according to the published protocol (Zou, Z., Najar, F., Wang, Y., Roe, B., Jiang, H., 2008. Pyrosequence analysis of expressed sequence tags for Manduca sexta hemolymph proteins involved in immune responses. Insect Biochem Mol Biol 38, 677-682). After shearing via nebulization, the four samples were end-repaired (Roe. B., 2004. Shotgun library construction for DNA sequencing. Methods Mol Biol 255: 171-187) and ligated to double-stranded adaptor A and biotinylated adaptor B (Margulies, M., Egholm, M., Altman, W.E., Bader, J.S. et al. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437 (7057), 376-380).