Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs
- Resource Type
- research-article
- Authors
- Erwin L. van Dijk, author; Evangelia Eleftheriou, author; Claude Thermes, author
- Source
- Journal of Visualized Experiments. (151)
- Subject
- 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
- Language
- English
- ISSN
- 1940-087X
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.