An improved protocol for small RNA library construction using High Definition adapters

Ping Xu, Martina Billmeier, Irina-Ioana Mohorianu, Darrell Green, William Fraser, Tamas Dalmay

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Next generation sequencing of small RNA (sRNA) libraries is widely used for studying sRNAs in various biological systems. However, cDNA libraries of sRNAs are biased for molecules that are ligated to adapters more or less efficiently than other molecules. One approach to reduce this ligation bias is to use a pool of adapters instead of a single adapter sequence, which allows many sRNAs to be ligated efficiently. We previously developed High Definition (HD) adapters for the Illumina sequencing platform, which contain degenerate nucleotides at the ligating ends of the adapters. However, the current commercial kits produced a large amount of 5’ adapter – 3’ adapter ligation product without the cDNA insert when HD adapters were used to replace the kit adapters. Here, we report a protocol to generate sRNA libraries using HD adapters with dramatically reduced adapter-adapter product. This protocol was developed from the procedure invented by Vaidyanathan et al. The libraries can be completed within two days and can be used for various biological and clinical samples. As examples for using this protocol, we constructed sRNA libraries using total RNA extracted from cultured mammalian cells and plant leaf tissue. The PCR products contained a very small amount of adapter-adapter product. Bioinformatic analysis of the sequencing data revealed sRNAs with diverse sequences and many different miRNA families.
Original languageEnglish
JournalMethods in Next Generation Sequencing
Issue number1
Publication statusPublished - 2 Apr 2015


  • RNA-seq
  • reduction of ligation bias
  • Medicago truncatula
  • Chondrosarcoma cell line

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