Defining pervasive transcription units using chromatin RNA-sequencing data

• Published in: STAR protocols Vol. 3; no. 2; p. 101442
• Main Authors: Guo, Ziwei, Liu, Xinhong, Chen, Mo
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17.06.2022; Elsevier
• Subjects: Bioinformatics; ChIPseq; CRISPR; Gene Expression; Genomics; Molecular Biology; Protocol; RNAseq; Sequence analysis; Sequencing; CRISPR; Gene Expression; Molecular Biology; Genomics; ChIPseq; Bioinformatics; Sequence analysis; Sequencing; RNAseq
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2022.101442

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). [Display omitted] •Efficient chromatin RNA extraction with spike-in RNA for RNA-seq normalization•Detection of accumulated PTs by chromatin RNA-seq upon Dis3 depletion•Annotate genome-wide PTs de novo•Bioinformatic pipeline for identification of sample-specific eRNAs and PROMPTs Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.