Common cell lysis procedures distort ribosome profiling analyses of gene expression

O'Connell, Aoife; Fedorova, Alla D; O'Connor, Patrick B F; Zhdanov, Alexander V; Baranov, Pavel V; Loughran, Gary; Andreev, Dmitry E

Common cell lysis procedures distort ribosome profiling analyses of gene expression

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s13059-025-03651-1.pdf(1.71 MB)

Published Version

Date

2025-08-11T00:00:00Z

Authors

O'Connell, Aoife

Fedorova, Alla D

O'Connor, Patrick B F

Zhdanov, Alexander V

Baranov, Pavel V

Loughran, Gary

Andreev, Dmitry E

Publisher

Springer Nature

Published Version

10.1186/s13059-025-03651-1

Abstract

Ribosome profiling is a powerful technique used to study gene expression on a transcriptome-wide scale. It involves sequencing of mRNA fragments protected by ribosomes from ribonuclease digestion. The initial steps commonly involve cell lysis followed by centrifugation and ribonuclease digestion. We find that centrifugation depletes 329 translated mRNAs in HEK293T cells. Many of these mRNAs encode cytoskeleton proteins. This suggests that the expression of a subset of mRNAs may be significantly underestimated in most ribosome profiling experiments. We show that omitting the centrifugation step after cell lysis can resolve this issue.

Keywords

Cytoskeleton , Gene expression , Insoluble fraction , Lysis , Nascent peptide , Polysome profiling , Riboseq , Ribosome profiling

Citation

O'Connell, A., Fedorova, A. D., O’Connor, P. B., Zhdanov, A. V., Baranov, P. V., Loughran, G. and Andreev, D. E. (2025) 'Common cell lysis procedures distort ribosome profiling analyses of gene expression', Genome Biology, 26(1), 241 (9pp). https://doi.org/10.1186/s13059-025-03651-1