Journal
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 151, Pages -Publisher
JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/60056
Keywords
Genetics; Issue 151; small RNA; small RNA-seq; bias; library preparation; next-generation sequencing; NGS; 2 '-O-methyl (2 '-OMe) RNA; plant microRNA; plant miRNA
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Funding
- National Center for Scientific Research (CNRS)
- French Alternative Energies and Atomic Energy Commission (CEA)
- Paris-Sud University
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The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the 'TruSeq (TS)' protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol 'TS5', which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.
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