Calibrated ribosome profiling assesses the dynamics of ribosomal flux on transcripts.

Ribosome profiling, which is based on deep sequencing of ribosome footprints, has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However, the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here, we overcome these hurdles through the development of "Ribo-FilterOut", which is based on the separation of footprints from ribosome subunits by ultrafiltration, and "Ribo-Calibration", which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript, the translation initiation rate, and the overall number of translation events before its decay, all in a genome-wide manner. Moreover, our method reveals the allocation of ribosomes under heat shock stress, during aging, and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome.