5 July

New sequencing technique maps total RNA in single cells

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Researchers from the Hubrecht Institute and the University of Cambridge have developed a novel technique with which they can map all RNA molecules in single cells. This work was recently published in the journal Nature Biotechnology.

Single-cell RNA sequencing allows researchers to determine which genes are switched on and which genes are switched off. This is measured based on the number of the different RNA molecules in the cell. Each of the RNA molecules ready for translation has a tail of a repeating base called adenine, or in short “A”. This tail of A’s, or polyA tail, allows researchers to recognize them, gain insight into the transcriptome of a cell and determine which genes are turned on and off.

However, although single-cell sequencing has become part of the standard repertoire of methods in the lab, the current techniques also have limitations. Only RNA molecules with a polyA tail are measured. That is only a fraction of all RNA molecules present in an individual cell.

Big picture
Scientists at the Hubrecht Institute and the University of Cambridge have developed a technique to detect all RNA molecules using single-cell sequencing analysis even if they do not have a polyA tail. For this technique, which they call Vast Transcriptome Analysis of Single cells by dA-tailing (VASA-seq), using a bacterial enzyme, they provide all RNA molecules in one cell with a polyA tail. These molecules can then be detected using the standard single-cell sequencing protocols. This way, VASA-seq can provide a snapshot of the big picture – all RNA molecules in a single cell, including those that are non-coding.

Higher resolution
This novel approach allows scientists to study all types of RNA in an individual cell. This includes the formation of histones of which the RNA does not have a polyA tail. These proteins play a role in DNA condensation. Splicing, the process in which RNA is cut into pieces before translation, can also be mapped using VASA-seq. In addition, the technique allows for a higher senstivity of sequencing, because the entire RNA transcript is measured. As a result, more RNA can be detected and analyzed.

The versatility of VASA-seq is groundbreaking, says Alexander van Oudenaarden, research leader and director at the Hubrecht Institute. ‘Previously, we could only measure a small part of the RNA. Now that we can measure the whole transcriptome, we can start gathering data on how cell types are being defined beyond polyA RNA molecules.’ At the Hubrecht Institute, VASA-seq is already being used as a standard method to analyze the genome of single cells, says Van Oudenaarden. ‘We are already combining VASA-seq with other analyses in the field of epigenetics and translation.

High-throughput total RNA sequencing in single cells using VASA-seq. Fredrik Salmen*, Joachim De Jonghe*, Tomasz S. Kaminski, Anna Alemany, Guillermo Parada, Joe Verity-Legg, Ayaka Yanagida, Timo N. Kohler, Nicholas Battich, Floris van den Brekel, Anna L. Ellermann, Alfonso Martinez Arias, Jennifer Nichols, Martin Hemberg, Florian Hollfelder & Alexander van Oudenaarden
DOI: 10.1038/s41587-022-01361-8
*these authors contributed equally

Image van Oudenaarden



Alexander van Oudenaarden is director of the Hubrecht Institute, group leader, professor of Quantitative Biology of Gene Regulation at the UMC Utrecht and Utrecht University and Oncode Investigator.