Transcriptome analysis of the Spodoptera exigua challenged with active or inactivated Autographa
The mRNA was purified from the total RNA extracted from 5th instar larvae of S. exigua using the Oligotex mRNA Midi Kit (QIAGEN, Germany). Then, double-stranded cDNA was synthesized using the SMART cDNA Library Construction Kit (Clontech, USA). PCR products of cDNA were further purified using the QIAquick PCR Purification Kit (QIAGEN, Germany) and checked for quality using the Agilent 2100 Bioanalyzer. Approximately 5 ug of cDNA was used for sequencing on a Roche/454 GS-FLX Titanium instrument (Roche, Germany). A 1/8-plate sequencing run was performed at MACROGEN Co. (Korea) following manufacturer's instructions. The raw 454 sequence files in SFF format were processed to remove low quality regions and adaptor sequences using programs SeqClean (http://compbio.dfci.harvard.edu/tgi/software). The resulting sequences were then screened against the NCBI UniVec database and E. coli genome sequences to remove possible contaminations of these sequences. Sequences shorter than 100 bp were discarded. The processed 454 sequences were assembled into unigenes using the iAssembler program (http://bioinfo.bti.cornell.edu/tool/iAssembler ).