A detailed protocol for iTP-Seq

Gillard, M. & Innis, C.A. (2024). iTP-Seq: a scalable profiling method to study context-dependent translational events in vitro. PROTOCOL (Version 1) available at Protocol Exchange.

We present a reproducible protocol for iTP-Seq, which substantially reduces the time and effort required to generate sequencing libraries. This protocol consists in 12 main steps: DNA library amplification, in vitro transcription, 5'-end mRNA biotinylation, in vitro translation, RNase R digestion, retention on streptavidin-coated beads, linker ligation, reverse transcription, second-strand synthesis, restriction enzyme digestion, PCR amplification and NGS adapter addition. The time required to complete a single round of iTP-seq is approximately 10 days.

iTP-Seq can be used to characterize any bacterial process involving ribosome pausing or stalling in vitro. For example, we have used it to characterize context-dependent translation inhibition by the antibiotic TcmX, or the mechanism of ermD induction by the antibiotics erythromycin and telithromycin.