A Small RNA-Seq Protocol with Less Bias and Improved Capture of 2′-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNAs such as plant microRNAs (miRNAs) carry a 2′-O-methyl (2′-OMe) modification at their 3′ terminal nucleotide. This modification adds another level of 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. We also provide guidelines for bioinformatics analysis of the sequencing data.