Preparation of Small RNA Libraries for High-Throughput Sequencing

Abstract

This protocol details the process of small RNA cloning for sequencing on the Illumina/Solexa sequencing platform, but it can be easily modified for use on other next-generation platforms (e.g., SOLiD, 454). This procedure is designed to clone canonical small RNA molecules with 5′-monophosphate and 3′-hydroxyl termini. Modifications, such as the presence of a 2′-O-methyl group, can reduce efficiency, although not sufficiently to negate the utility of the approach. Other termini modifications, such as a 5′ triphosphate or a 3′ phosphate, can be altered by enzymatic treatment before cloning.