Capped small RNA sequencing (csRNA-seq) for genome-wide mapping of active transcription start sites and enhancer RNAs from total RNA

Profiling ongoing transcription reveals the complete active transcriptome, including unstable enhancer RNAs, and provides a dynamic view of transcription dynamics, enabling the study of underlying gene regulatory mechanisms. Here, we describe a detailed protocol for capped- small RNA- sequencing (csRNA-seq), a method that uses total RNA to capture actively initiating or ‘nascent’ RNA polymerase II transcripts, including unstable RNAs such as enhancer RNAs, and their transcription start sites (TSSs). csRNA-seq offers several advantages: (1) the decoupling of sample collection and processing; (2) compatibility with diverse sample types, including fresh, frozen, and fixed tissues, as well as various eukaryotic organisms; and (3) scalability. Purified RNA is further non-infectious, can be isolated from inactivated samples—including clinical specimens or pathogenic samples—transported, and studied under standard laboratory conditions. This accessible protocol empowers researchers with minimal experience in nascent transcriptomics to study gene regulation and transcription dynamics. The protocol involves small RNA isolation, 5′-capped RNA enrichment, and library generation, followed by sequencing and data analysis to compare csRNA-seq and input small RNA to identify nascent RNAs.