QsRNA-seq: a method for high-throughput profiling and quantifying small RNAs

Abstract

The finding that small non-coding RNAs (sRNAs) can affect cellular processes by regulating gene expression had a significant impact on biological research and clinical diagnosis. Yet, the ability to quantify and profile sRNAs, specifically miRNAs, using high-throughput sequencing is especially challenging because of their small size and repetitive nature. We developed QsRNA-seq, a method for preparation of sRNA libraries for high-throughput sequencing that overcomes this difficulty by enabling separation of fragments shorter than 100nt long that differ only by 20nt in length. The method supports using unique molecular identifiers for quantification. We show that QsRNA-seq gives very accurate, comprehensive and reproducible results. Using QsRNA-seq to study the miRNA repertoire in C. elegans embryo and L4 larval developmental stages, enabled extending the list of miRNAs that are expressed in a developmental-specific manner. Interestingly, we found that miRNAs 23nt long are predominantly expressed in developmental stage L4, suggesting a possible connection between the length of miRNA and its developmental role.