SummarySequencing chromatin-associated RNA using libraries from the chromatin fraction makes it possible to characterize RNA processing driven by disassociated subunits. Here, we present an experimental strategy and computational pipeline for processing chromatin-associated RNA-seq data to detect and quantify readthrough transcripts. We describe steps for constructing degron mouse embryonic stem cells, detecting readthrough genes, data processing, and data analysis. This protocol can be adapted to various biological scenarios and other types of nascent RNA-seq, such as TT-seq.For complete details on the use and execution of this protocol, please refer to Li et al. (2023).1