Increasing Ligation Efficiency and Reducing Bias for Small RNA Library Prep with Plant Samples

MicroRNAs (miRNAs) are 18-22 nucleotide long non-coding small RNAs that regulate protein expression involved in various cellular process such as development, growth, and physiology. Many studies have shown that miRNA expression in plants is altered by stress-response and environmental changes. Thus, the analysis of miRNA profiles using next generation sequencing can be beneficial to our understanding of stress tolerance of crops and other plants. Although miRNAs in plants and animals share many similarities, most mature miRNAs in plants contain 2’-O-methylation at the 3’ end. Typically the 3’ adapter ligation is the first step in small RNA library construction. 2’-O-methylation of plant miRNAs reduces ligation efficiency of the 3’ OH, making plant miRNA libraries difficult to generate. Here, we show that miRNA profiling from corn and rice using total RNA inputs can be successfully achieved using the NEXTflex™ Small RNA Sequencing Kit v3, without library purification from PAGE gels. Extending the ligation incubation step increases library yield. Importantly, many putative novel miRNAs were discovered with the NEXTflex™ Small RNA Sequencing Kit v3 that were not discovered by other methods. Thus, this protocol allows generation of reduced-bias small RNA libraries from plant total RNA without the tedious step of gel-based size selection, enabling researchers to discover and profile more small RNAs from more samples than with traditional methods.