ABC-seq expands small RNA content with randomized adapter pool editing

Cellular gene expression is broadly regulated by a large number of small RNAs (smRNAs). The ability to deeply analyze the content of smRNAs is vital for a comprehensive understanding of their architecture and function. However, experimental approaches that can realize both high-content and low-byproduct sequence analyses are lacking. Here, we develop ABC-seq (randomized Adapter pool dimer Blocking and Cleavage in smRNA sequencing). This method designs randomized adapter pools to recognize more smRNA species. Moreover, it performs two product structure-differentiated enzymatic reactions to maximally eliminate the byproducts of adapter dimers. Using ABC-seq, we detected a wider range of smRNAs, including miRNAs, scRNAs, and snoRNAs in rat hypertrophic cardiomyocytes. After further analysis of miRNAs, we detected 66 more miRNAs compared with the control method, which provides new insights into smRNA-driven gene regulation mechanisms. Furthermore, we have revealed cancer drug response-associated smRNAs and uncovered their crucial role in immune response modulation in non-small cell lung cancer. ABC-seq represents an evolutionary strategy to reshape the boundaries of the smRNA landscape and paves the way for a deeper understanding of complex gene regulatory networks.