NET-CAGE characterizes the dynamics and topology of human transcribed cis-regulatory elements

Promoters and enhancers are key cis-regulatory elements, but how they operate to generate cell type-specific transcriptomes is not fully understood. We developed a simple and robust method, native elongating transcript–cap analysis of gene expression (NET-CAGE), to sensitively detect 5′ ends of nascent RNAs in diverse cells and tissues, including unstable transcripts such as enhancer-derived RNAs. We studied RNA synthesis and degradation at the transcription start site level, characterizing the impact of differential promoter usage on transcript stability. We quantified transcription from cis-regulatory elements without the influence of RNA turnover, and show that enhancer–promoter pairs are generally activated simultaneously on stimulation. By integrating NET-CAGE data with chromatin interaction maps, we show that cis-regulatory elements are topologically connected according to their cell type specificity. We identified new enhancers with high sensitivity, and delineated primary locations of transcription within super-enhancers. Our NET-CAGE dataset derived from human and mouse cells expands the FANTOM5 atlas of transcribed enhancers, with broad applicability to biomedical research.