Multiple aspects of mRNA translation are subject to regulation. Here we present a ribosome footprinting protocol to determine the location and composition of 40S and 80S ribosome complexes on endogenous mRNAs transcriptome-wide in vivo in yeast and mammalian cells. We present an extension of the translation complex profiling (TCP-seq) protocol, originally developed in yeast, by including an immunoprecipitation step to assay the location of both 40S and 80S ribosome complexes containing proteins of interest. This yields information on where along mRNAs the ribosome-bound protein of interest joins the ribosome to act, and where it leaves again, thereby monitoring the sequential steps of translation and the roles of various translation factors therein. Rapid fixation of live cells ensures the integrity of all translation complexes bound to mRNA at native positions. Two procedures are described, differing mainly in the fixation conditions and the library preparation. Depending on the research question, either procedure offers advantages. Execution of a Sel-TCP-seq experiment takes 5-10 working days, and initial data analysis can be completed within 2 days.

German Cancer Research Center Heidelberg Germany

Heidelberg University Heidelberg Germany

Laboratory of Bioinformatics Institute of Microbiology CAS Prague Czech Republic

Laboratory of Regulation of Gene Expression Institute of Microbiology CAS Prague Czech Republic

Max Planck Institute of Molecular Cell Biology and Genetics Dresden Germany

Shine Dalgarno Centre for RNA Innovation and Division of Genome Sciences and Cancer John Curtin School of Medical Research Australian National University Canberra Australia

University of Paris Imagine Institute Paris France

Victor Chang Cardiac Research Institute Darlinghurst Australia

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