MicroRNAs and the Immune System pp 93-111
Construction of Small RNA cDNA Libraries for Deep Sequencing
Since the phenomenon of small RNA-mediated gene silencing was first described over 15 years ago (Lee et al. Cell 75:843–854, 1993; Wightman et al. Cell 75:855–862, 1993), it has become evident that a variety of endogenous small RNAs play an important role in establishing and maintaining cell lineages. MicroRNAs (miRNAs), in particular, have been shown to exert regulatory control over the development and function of the many specialized cells that comprise the mammalian immune system (Baltimore et al. Nat Immunol 9:839–845, 2008; Kanellopoulous and Monticelli Semin Cancer Biol 18:79–88, 2008; Xiao and Rajewsky Cell 136:26–36, 2009). The advent of next generation sequencers provides an important tool for profiling the small RNA transcriptome of many diverse cell types. Compared to traditional Sanger sequencing, next generation sequencing machines can process millions of sequence reads in parallel, generating megabases of data within just a few days. The generation of small RNA libraries for sequencing is relatively straightforward and involves the ligation of platform-specific adapter sequences to small RNAs, followed by reverse transcription of the ligated species and PCR amplification. While other hybridization-based techniques are available for profiling well-characterized small RNAs, high-throughput sequencing remains the most powerful method for discovering novel small RNAs and posttranscriptional editing.