ABC-seq expands small RNAs content with randomized adapter pools operation

Abstract

AbstractCellular gene expression is widely regulated by a large number of small RNAs (smRNAs). The ability to in-depth analysis of smRNAs content is vital for comprehensive understanding their architecture and function. However, experimental approaches capable of realizing both high-content and low-byproduct sequencing analysis are lacking. Here, we develop ABC-seq (randomizedAdapter pool dimerBlocking andCleavage in smRNA sequencing). This method designs randomized adapter pools to recognize more smRNA species. Moreover, it operates two product structure-differentiated enzymatic reactions to maximally eliminate the byproducts of adapter dimers. Using ABC-seq, we detected a broader range of smRNAs including miRNAs, scRNAs, and snoRNAs in rat hypertrophic cardiomyocytes. Upon further analysis of miRNAs, we detected 66 more miRNAs compared to the control method, providing new insights into smRNA-directed gene regulation mechanisms. Furthermore, we revealed cancer drug response -associated smRNAs and uncovered their pivotal role in immune response modulation in non-small cell lung cancer. ABC-seq represents an evolutionary strategy for reshaping the smRNA landscape boundaries and paves the way for a deeper understanding of complex gene regulation networks.