Popillia japonica antennal transcriptome
Library preparations for GS Jr (Roche/454 Life Sciences) were developed by the primary author by integrating cDNA synthesis, normalization, adapter ligation, and emulsion PCR and are available upon request.  Briefly, cDNA was synthesized from 1200 ng of pooled total antenna RNA in a manner similar to Clontech SMART protocols, using primers optimized for the 454 sequencing process and amplified by PCR to generate dsDNA.  The cDNA library was normalized using the Trimmer cDNA normalization kit (Evrogen) according to the manufacturer’s instructions.  Normalized DNA was then fragmented by sonication, and ends were enzymatically blunted and ligated to 454 adaptors.  Amplification of ligation products exploited adaptor-mediated PCR suppression.  The procedure induces homo-mediated fragment hairpins, thereby severely limiting amplification of mis-ligated products.  All amplification steps utilized high-fidelity polymerases.  Emulsion PCR reactions were performed according to the manufacturer (Roche/454 Life Sciences).  To optimize the pyrosequencing throughput, the final libraries were titrated by emulsion PCR bead enrichment prior to sequencing.  Sequencing of the antennae cDNA library was performed on a picotitre plate according to the manufacturer’s instructions.  Sequencing adaptors were automatically removed from the reads using signal processing software (Roche/454 Life Sciences).