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bioRxiv · 10.1101/2025.03.07.642103

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

Eastman, G.·Bloom, G. S.·Sotelo Silveira, J. R.
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Abstract

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 that exhibit expected characteristics in size, transcriptome mapping, and periodicity. Comparing translatome from Benzonase- and RNAse I-derived footprints reveals minimal differences underscoring Benzonases potential to streamline the protocol, reducing time, cost and bias. 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. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/642103v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@b5e325org.highwire.dtl.DTLVardef@153ccf9org.highwire.dtl.DTLVardef@b5f123org.highwire.dtl.DTLVardef@ea6bab_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Eastman, G., Bloom, G. S., Sotelo Silveira, J. R.. 2025-03-11. The use of Benzonase to produce ribosome footprints simplifies translational levels quantification by Ribo-seq. https://doi.org/10.1101/2025.03.07.642103

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