Common cell lysis procedures distort ribosome profiling analyses of gene expression
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.
Authors
- Alexander V. Zhdanov (ORCID: https://orcid.org/0000-0001-5787-2035)
- Pavel V. Baranov (ORCID: https://orcid.org/0000-0001-9017-0270)
- Dmitry E. Andreev (ORCID: https://orcid.org/0000-0001-5320-3045)
- Alla D. Fedorova (ORCID: https://orcid.org/0000-0001-5996-7855)
- Patrick B. F. O’Connor (ORCID: https://orcid.org/0000-0003-1085-2795)
- Aoife O’Connell (ORCID: https://orcid.org/0000-0003-2577-4937)
- Gary Loughran
Institutions
- University College Cork (IE)
- Institute of Bioorganic Chemistry (RU)
- Bio-Medical Science (South Korea) (KR)
- Yandex (Russia) (RU)
Publication Details
- Journal
- Genome biology
- Published
- 2025-08-11
- DOI
- https://doi.org/10.1186/s13059-025-03651-1
- Citations
- 1
- Primary Topic
- RNA and protein synthesis mechanisms
- Type
- article
- Field-Weighted Citation Impact
- 0.81
Funders
- Science Foundation Ireland
- Irish Research Council
- Russian Science Foundation