A Computational Pipeline for Intergenic/Intragenic Enhancer RNA Quantification in Mouse Embryonic Stem Cells

Core cis-regulatory elements known as enhancers play a central role in enabling precise transcriptional regulation of target genes that control diverse cellular functions and developmental processes. These enhancers are often transcribed in both directions, producing long non-coding transcripts referred to as enhancer RNAs (eRNAs). The expression of eRNAs is closely linked to active chromatin features, such as H3K27ac and co-activator recruitment, and functionally contributes to the transcriptional activation of target genes. Nevertheless, the detection and quantification of eRNAs remain challenging, especially when they overlap with host-gene transcription. To address this, we present a standardized, user-friendly computational workflow for analyzing enhancer transcription from nascent RNA sequencing data. The protocol guides users through data preprocessing, read mapping, and quality control, followed by strand-specific quantification of enhancer-associated transcription, with dedicated procedures for intragenic enhancers where signal assignment is complex. Visualization modules enable clear inspection of enhancer activity across genomic contexts, and built-in options support analyses of both intergenic and intragenic enhancers. Designed for researchers with limited bioinformatics expertise, this workflow provides a practical framework for consistent, reproducible, and scalable studies of enhancer transcription, facilitating broader application of enhancer biology across diverse systems.