Azidonucleoside-incorporated RNA sequencing for RNA stability analysis in E.coli.

To study RNA degradation by RNA-seq, global inhibition of transcription has to be performed in order to measure RNA decay with no interference from RNA sysnthesis. However, transcription inhibitors are generally toxic, can affect abundance many transcripts, and cause delay in RNA decay by residual RNA synthesis. We envisioned that AIR-seq, in conjunction with the pulse-chase labeling, would enable genome-wide analysis of RNA degradation in E. coli with no need of transcription inhibition. Overall design: After pulse labeling with AzG for 2 h, the E. coli cells were chased with guanosine for 0, 5, and 10 min, respectively, followed by extraction of total RNA. After reacting with DBCO-biotin, the labeled RNA were isolated by streptavidin beads and subjected to RNA-seq.