Small RNA library cloning procedure for deep sequencing of specific endogenous siRNA classes in Caenorhabditis elegans

Abstract

In recent years, distinct classes of small RNAs ranging in size from ~21 to 26 nucleotides have been discovered and shown to play important roles in a wide array of cellular functions. Because of the abundance of these small RNAs, library preparation from an RNA sample followed by deep sequencing provides the identity and quantity of a particular class of small RNAs. In this chapter we describe a detailed protocol for preparing small RNA libraries for deep sequencing on the Illumina platform from the nematode C. elegans.

Fig. 1

The three major classes of sRNAs in C. elegans. (a) miRNAs (~21–22 nucleotides long) are Dicer-dependent and thus have a 5′ monophosphate and a 3′ hydroxyl modifications. Most (>70 %) miRNAs start with a uridine at the 5′ end (indicated as N*).The ~30 % of the remaining miRNAs start with an adenosine, cytidine, or guanosine. (b) 21U-piRNAs exhibit a 5′ monophosphate uracil and a 2′-methyl moiety at the 3′ end. (c) Endo-siRNAs are categorized as primary endo-siRNAs (26G-siRNAs) and secondary endo-siRNAs (22G-siRNAs). The lesser abundant 26G-siRNAs are Dicer-dependent and have a 5′ monophosphate guanosine and a 2′,3′ hydroxyl group at the 3′ end. The more abundant 22G-siRNAs are Dicer-independent, RdRP-dependent, and have a 5′ triphosphate guanosine and a 3′ hydroxyl group. The use of TAP (see Subheading 3.1) allows for the conversion of 5′ triphosphate to a monophosphate group required for sRNA library cloning following this protocol. G* indicates that over 90 % of endo-siRNAs have a guanosine at the 5′ end. The remaining ~10 % of endo-siRNAs can start with an adenosine, cytidine, or uridine

Fig. 2

Flowchart of sRNA library synthesis. This flowchart illustrates the steps of sRNA library synthesis outlined in this chapter. The numbers indicate which step in the protocol is being illustrated. Illustrative examples of polyacrylamide gels used for sRNA library size selection after completion of (a) Subheading 3.2 3′ adapter ligation and (b) Subheading 3.4 reverse transcription and library amplification steps are shown. The dotted lines indicate the regions of the gels that were extracted. Asterisk indicates the primer dimer artifact described in the text