Preparation of small RNA libraries for high-throughput sequencing

Cold Spring Harb Protoc. 2012 Oct 1;2012(10):1067-77. doi: 10.1101/pdb.prot071431.


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.

MeSH terms

  • Cloning, Molecular / methods*
  • Gene Library
  • High-Throughput Nucleotide Sequencing / methods*
  • RNA / genetics
  • RNA / isolation & purification*


  • RNA