ABC‐seq Expands Small RNAs Profiling with Successive Nucleic Acid Structure‐Differentiated Enzymatic Recognition

Abstract Small RNAs (smRNAs) play crucial roles in gene regulation, but current sequencing methods face a set of tangled challenges, including adapter ligation bias, high adapter dimer byproducts, and amplification variability, which limit their capacity to fully capture smRNA diversity. Here, we develop ABC‐seq, a general strategy to enhance smRNA recognition through successive structure‐differentiated recognition. We designed two randomized adapters, each containing a restriction enzyme recognition motif or a cleavage site. Then, we operate two product structure‐differentiated enzymatic reactions to maximally suppress randomized adapter dimer. Furthermore, the randomized sequence in adapters serves as unique molecular identifiers (UMIs) in smRNA‐seq data analysis to correct amplification bias and improves quantification accuracy. Using ABC‐seq, we identified a wider range of smRNAs, including miRNAs, scRNAs, and snoRNAs, in hypertrophic cardiomyocytes, detecting 9.4% more miRNAs compared to traditional methods. Additionally, ABC‐seq revealed smRNAs associated with cancer drug responses and their roles in immune modulation in non‐small cell lung cancer. ABC‐seq expands the smRNA landscape and offers new insights into complex gene regulatory networks.