Small RNA Profiling by Next-Generation Sequencing Using High-Definition Adapters

  • Martina Billmeier
  • Ping XuEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1580)


Small RNAs (sRNAs) as key regulators of gene expression play fundamental roles in many biological processes. Next-generation sequencing (NGS) has become an important tool for sRNA discovery and profiling. However, NGS data often show bias for or against certain sequences which is mainly caused by adapter oligonucleotides that are ligated to sRNAs more or less efficiently by RNA ligases. In order to reduce ligation bias, High-definition (HD) adapters for the Illumina sequencing platform were developed. However, a large amount of direct 5′ and 3′ adapter ligation products are often produced when the current commercially available kits are used for cloning with HD adapters. In this chapter we describe a protocol for sRNA library construction using HD adapters with drastically reduced direct 5′ adapter–3′ adapter ligation product. The protocol can be used for sRNA library preparation from total RNA or sRNA of various plant, animal, insect, or fungal samples. The protocol includes total RNA extraction from plant leaf tissue and cultured mammalian cells and sRNA library construction using HD adapters.

Key words

Small RNA Next-generation sequencing Small RNA profiling Library construction Reduction of ligation bias 


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© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.School of Biological SciencesUniversity of East AngliaNorwichUK

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