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Summary: Pervasive transcripts (PTs) are difficult to detect by steady-state RNA-seq, because they are degraded immediately by the nuclear exosome complex. Here, we describe a protocol illustrating a bioinformatic pipeline for genome-wide PTs de novo annotation via chromatin-associated RNA-seq data upon DIS3 depletion. Compared to defining PTs by nascent RNA-seq such as TT-seq and PRO-seq, this protocol is more convenient and cost efficient. In addition, this protocol defines 3′-end of PTs more precisely, while reads from PRO-seq have a skew at the 5′-end.For complete details on the use and execution of this protocol, please refer to Liu et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.