Quantifying RNA synthesis at rate-limiting steps of transcription using nascent RNA-sequencing data

• Published in: STAR protocols 2022-03, Vol.3 (1), p.101036-101036, Article 101036
• Main Authors: Rabenius, Adelina, Chandrakumaran, Sajitha, Sistonen, Lea, Vihervaara, Anniina
• Format: Article
• Language: English
• Subjects: Base Sequence; Bioinformatics; Chromatin immunoprecipitation (ChIP); Gene Expression; Genetic; Genetics; Genomics; human; Humans; Nucleic Acid; nucleotide sequence; procedures; promoter region; Promoter Regions; Promoter Regions, Genetic; Protocol; regulatory sequence; Regulatory Sequences; Regulatory Sequences, Nucleic Acid; RNA; RNA - genetics; RNAseq; sequence analysis; Sequence Analysis, RNA - methods; Systems biology
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.101036

Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide analytical steps to (1) identify transcribed regulatory elements de novo genome-wide, (2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies, and termination windows, and (3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol tracks engaged transcription complexes across functional genomic regions demonstrated in human K562 erythroleukemia cells. For complete details on the use and execution of this protocol, please refer to Vihervaara et al. (2021). [Display omitted] •Identification of transcribed regulatory elements de novo genome-wide•Quantification of engaged transcription complexes at functional genomic regions•Measuring distribution of transcription regulators across the functional genomic regions•Revealing functional genomic regions from nascent transcription data Nascent RNA-sequencing tracks transcription at nucleotide resolution. The genomic distribution of engaged transcription complexes, in turn, uncovers functional genomic regions. Here, we provide analytical steps to (1) identify transcribed regulatory elements de novo genome-wide, (2) quantify engaged transcription complexes at enhancers, promoter-proximal regions, divergent transcripts, gene bodies, and termination windows, and (3) measure distribution of transcription machineries and regulatory proteins across functional genomic regions. This protocol tracks engaged transcription complexes across functional genomic regions demonstrated in human K562 erythroleukemia cells.