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

The finding that small non-coding RNAs (sRNAs) can affect cellular processes by regulating gene expression had a significant impact on biology research and clinical diagnosis. Yet, the ability to quantify and profile sRNAs, especially miRNAs, using high-throughput sequencing is especially challenging because of their repetitive nature. We have developed QsRNA-seq a method for preparation of sRNA libraries for high-throughput sequencing that overcomes this difficulty by enabling separation of fragments differing only by 20nt in length and implementing barcode and unique molecular identifiers for multiplexing and accurate quantification. We show that this method gives very accurate, comprehensive and reproducible results. Using QsRNA-seq to study the miRNA repertoire in C. elegans embryo and L4 larval developmental stages, we found many miRNAs that are expressed in a developmental-specific manner. Interestingly, when profiling miRNA length, we found that miRNAs 23-nt long are predominantly expressed in L4 developmental stage and not embryo. RNA was extracted from wildtype C. elegans synchronized to embryo or L4 larval stage. For each stage, 3 independent biological replica RNA samples were used for construction of small RNA libraries. One of the 3 samples was also used for preparation of three technical replica libraries and, in addition, for preparation of 3 technical replicas of libraries without UMI (0N) at 5-adaptor. One library was prepared from human brain total RNA (Life technologies).