Small non-coding RNAs play a vital regulatory role in cells (reviewed in 1
). The most abundant small RNAs in animals are 20 to 23 nucleotide (nt) long microRNAs (miRNAs). The first miRNA members were identified in C. elegans
). The discovery that double-stranded RNA (dsRNA) triggered RNA interference (RNAi) (10
) was also mediated by similar-sized small RNA processing products, known as small interfering RNAs (siRNAs) (11
), prompted the development of techniques to characterize naturally occurring small RNAs (15
). These methods were based on small RNA cDNA library preparation and sequencing and ignited the discovery of new members and families of small RNAs (15
Small RNAs, in association with their protein effector components, mediate sequence-specific posttranscriptional and transcriptional gene regulation. They control mRNA translation, stability and localization (reviewed in 25
) and feed into processes that control transposons (reviewed in 27
) and heterochromatin structure (reviewed in 4
). This wide range of functions stimulated great interest to identify and characterize the small RNAs expressed in different organisms, tissues and cell types, in normal and disease states.
Here we describe our protocols for the construction of small RNA libraries and their adaptation for various high throughput sequencing approaches. The protocols originate from methods described previously (30
) and provide new details regarding the use of RNA ligases and the latest sequencing technology.