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 overcomeovercame 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 revealed 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. Overall design: Ribosome profiling and RNA-seq of multiple cell lines to detect the global translation of each conditions. UPDATE: [Jun-18-2025] In the titles and processed data file names for Samples GSM7430255 and GSM7430256, "DMSO" was replaced by "RocA3uM". In the titles and processed data file names for Samples GSM7430259 and GSM7430260, "RocA3uM" was replaced by "DMSO". The files associated with each GSMxxx did not change.