Optimized Quantitative Assessment of Enhancer RNA Stability in Mouse Embryonic Stem Cells

Enhancer RNAs (eRNAs) are non-coding transcripts produced from transcriptionally active enhancer regions and are increasingly recognized as key regulators of gene expression. Despite their functional importance, the stability and degradation dynamics of eRNA remain poorly characterized, particularly in embryonic stem cells (ESCs), where transcriptional programs are highly dynamic and context-dependent. This protocol presents a quantitative method to assess the decay kinetics and half-life of intergenic and intragenic eRNAs in mouse ESCs. Transcription is inhibited using Actinomycin D, and total RNA is collected at multiple defined time points. cDNA is synthesized using both random hexamer and oligo(dT) primers to enable detection of both polyadenylated and non-polyadenylated transcripts. For intergenic eRNAs, normalization is performed using either a housekeeping gene or the Ct value at time 0 min. For intragenic eRNAs, a dual normalization approach is employed to minimize interference from overlapping pre-mRNA. The decay curves are analyzed using a one-phase exponential decay model, enabling accurate estimation of the half-life. The protocol reveals that eRNAs in mESCs exhibit rapid turnover, with a half-life of approximately 2-3 min. This methodology provides a robust and reproducible framework for studying eRNA stability, offering insight into enhancer activity and RNA dynamics in pluripotent stem cell contexts.