Large-Scale Sequencing of Plant Small RNAs

Deep sequencing technologies have become very powerful tools in the identification and quantification of small RNAs involved in gene regulation. Small interfering RNA (siRNA) and miRNA are two classes of DCL-dependent small RNAs known to affect phenotype, developmental regulation, and various traits in plants. These small RNAs function by selectively repressing gene expression mainly by guiding cleavage, resulting in degradation of target transcripts. In this chapter, we describe a method for preparation of 5^′ -phosphate-dependent small RNA libraries, a hallmark of RNase III-like DCL products, for high-throughput sequencing, and recommendations for small RNA analysis. This method is useful for determining small RNA involvement in critical pathways in plants, identifying and quantifying novel small RNAs, and examining small RNA global expression patterns.