High-resolution profiling of bacterial transcriptomes by SEnd-seq

Transcription-the process by which genomic DNA is converted into RNA-is a highly dynamic and tightly controlled process across all domains of life. Although bacteria were once regarded as relatively simple organisms, their transcriptomes are now recognized to be remarkably complex, heterogeneous, and subject to multilayered regulation. Despite the availability of an abundance of sequenced bacterial genomes, a comprehensive understanding of how bacteria tune their transcriptional output to adapt to changing environments remains lacking. To this end, SEnd-seq (simultaneous 5' and 3' end sequencing) was developed as a high-throughput approach uniquely capable of simultaneously capturing both 5' and 3' ends of individual RNA molecules, enabling the reconstruction of full-length transcripts. By capturing each RNA molecule as a distinct molecular entity with single-nucleotide resolution, SEnd-seq has uncovered previously unrecognized transcriptional features across diverse bacterial species, including even the well-studied Escherichia coli. This method performs robustly across a wide range of RNA species and organisms, including hard-to-lyse pathogens such as Mycobacterium tuberculosis. Moreover, SEnd-seq exhibits high sensitivity for detecting low-abundance RNA and is compatible with various target RNA enrichment strategies, as well as genetic, chemical, and functional perturbations, enabling context-specific transcriptomic analyses. As a versatile and broadly adaptable technology, SEnd-seq provides comprehensive insights into transcriptional regulation, RNA processing, and the coordination between RNA-based processes, thereby uncovering potential targets for antibiotic development. In the present review, we summarize the features and applications of SEnd-seq and discuss its future methodological development and expansion into broader biological and biomedical research contexts.