The use of Benzonase to produce ribosome footprints simplifies translational levels quantification by Ribo-seq.

Gene expression quantification through genomics methods is crucial for understanding diverse biological contexts. Among these methods, ribosome profiling (Ribo-seq) stands out as a valuable tool for uncovering post-transcriptional gene expression regulation by providing a comprehensive view of the translatome. While current protocols are time-intensive with limited variations, we introduced the use of the Benzonase enzyme to generate ribosome footprints from a polysome-enriched fraction, streamlining the workflow and reducing time, cost and potential sources of bias. Comparing translatome from Benzonase- and RNAse I-derived footprints reveals minimal differences, with both separating from RNA-seq-based transcriptome quantification. Benzonase acts more gently on ribosomes, yielding larger footprints and a weaker triplet periodicity compared to RNAse I, while enabling its application to low-input samples. We further demonstrate Ribo-seq application in primary neuronal cultures, using Benzonase to digest the post-mitochondrial supernatant, thereby bypassing the labor-intensive ribosome/polysome purification step. The introduction of such protocol variations for Ribo-seq, especially for challenging low-input samples, offers a significant advancement of this resource for the community.