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Next-generation sequencing technologies have allowed new tools to be developed for transcriptome characterization. We describe here the methods for the preparation of a library from pooled RNAs for 454 sequencing of 3'-cDNA fragments. We also describe how the read sequences obtained can be used to generate, through de novo reads assembly, a large catalog of unique transcripts in organisms for which a comprehensive collection of transcripts or the complete genome sequence is not available. Finally, this catalog can be used efficiently to design oligonucleotide probes for setting up a comprehensive microarray for global analysis of gene expression.