Abstract
Ribosome profiling is a powerful technique to discover translation events, and to reveal translational dynamics at gene and codon level. The procedure includes laborious ribosome isolation steps using density gradient ultracentrifugation. However, ultracentrifuges are not available in every laboratory, and ultracentrifugation is not suitable for high-throughput experimentation. PEG precipitation has been used to purify ribosomes and in this study we have tested whether PEG precipitation can be used as an alternative method to isolate ribosomes for ribosome profiling. We have benchmarked PEG precipitation against spin column-based size-exclusion and standard sucrose density ultracentrifugation methods for ribosome isolation. The methods were tested using the bacterial model system Bacillus subtilis treated with mupirocin, which stalls ribosomes at isoleucine codons, providing a testable reference for ribosome profile comparison. We found that the use of PEG precipitation, size-exclusion spin columns, and a combination of both, yielded ribosome profiles that were comparable to the standard procedure. With these methods, ultracentrifugation can be omitted, facilitating ribosome profiling and enabling high-throughput analyses.