Li-BrU-seq: A Low-Input and Simplified Metabolic Labeling Method for Nascent RNA Sequencing

Precise regulation of transcriptional dynamics underlies gene expression programs, governing critical biological processes such as cell fate determination, tissue development, and stress responses. While nascent RNA sequencing technologies offer powerful tools for dissecting these mechanisms, existing methods remain constrained by complex workflows, high cellular input requirements, and cytotoxicity. Here, we present Li-BrU-seq, a systematically optimized 5-bromouridine (BrU)-based profiling strategy designed for low-input samples. Rigorous benchmarking demonstrates that Li-BrU-seq outperforms previous protocols in both enrichment specificity and sensitivity. By streamlining the enrichment workflow, the method enables high-quality transcriptomic profiling from low-input material (500 ng total RNA or ∼25,000 cells). Furthermore, Li-BrU-seq supports flexible temporal resolution ranging from ultrashort pulses to long-term tracking, free from the stress-induced artifacts inherent to 4sU. Additionally, it offers tailored workflows compatible with diverse downstream applications. Li-BrU-seq provides an accessible and versatile platform that expands nascent RNA analysis to low-input, rare, and physiologically sensitive biological systems.