EU-RNA-seq for in vivo labeling and high throughput sequencing of nascent transcripts

Abstract

The protocol allows for labeling nascent RNA without isolating nuclei. The cell-permeable uridine analog, 5-ethynyluridine (EU), is added to media to allow in vivo labeling of nascent transcripts. Cells are lysed, total RNA is collected, and biotin is conjugated to EU-labeled RNAs. Custom biotin RNAs are added and biotinylated RNAs are isolated for generation of cDNA libraries. The sequencing data are normalized to controls for quantitative assessment of the nascent transcriptome. The protocol takes 4 days, not including sequencing and analysis. For complete details on the use and execution of this protocol, please refer to Palozola et al. (2017).

Topics & Concepts