Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are non-coding regulatory RNAs that play an important role in development and genome stability in plants. Conventional cloning and sequencing approaches have identified hundreds of miRNAs and a large number of siRNAs, but are no longer the best choices for identification of new miRNAs which are generally expressed at low abundance. The development of next-generation sequencing technologies has provided a powerful platform for the discovery of these small but vital RNA molecules. This chapter describes a protocol for the construction of small RNA cDNA libraries suitable for sequencing-by-synthesis (SBS) technologies, such as the Roche Genome Sequencer FLX and the Illumina Genome Analyzer platforms, which have been widely used to identify new miRNAs and other types of small RNAs.
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