Using GRO-Seq to Measure Circadian Transcription and Discover Circadian Enhancers

Methods Mol Biol. 2021:2130:127-148. doi: 10.1007/978-1-0716-0381-9_10.

Abstract

Circadian gene transcription transmits timing information and drives cyclic physiological processes across various tissues. Recent studies indicate that oscillating enhancer activity is a major driving force of rhythmic gene transcription. Functional circadian enhancers can be identified in an unbiased manner by correlation with the rhythms of nearby gene transcription.Global run-on sequencing (GRO-seq) measures nascent transcription of both pre-mRNAs and enhancer RNAs (eRNAs) at a genome-wide level, making it a unique tool for unraveling complex gene regulation mechanisms in vivo. Here, we describe a comprehensive protocol, ranging from wet lab to in silico analysis, for detecting and quantifying circadian transcription of genes and eRNAs. Moreover, using gene-eRNA correlation, we detail the steps necessary to identify functional enhancers and transcription factors (TFs) that control circadian gene expression in vivo. While we use mouse liver as an example, this protocol is applicable for multiple tissues.

Keywords: Circadian; Enhancer RNA; Global run-on sequencing; Nascent RNA; Transcription factor.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Circadian Rhythm Signaling Peptides and Proteins / genetics*
  • Circadian Rhythm Signaling Peptides and Proteins / metabolism
  • Enhancer Elements, Genetic / genetics*
  • Mice
  • Mice, Inbred C57BL
  • RNA, Small Untranslated / chemistry
  • RNA, Small Untranslated / genetics*
  • Sequence Analysis, RNA / methods*
  • Transcriptome

Substances

  • Circadian Rhythm Signaling Peptides and Proteins
  • RNA, Small Untranslated