Mie University and collaborators develop new single-cell technique for simultaneous DNA and RNA analysis

A joint research group including Professor Shin-ichiro Takebayashi, Assistant Professor Rawin Poonperm (RIKEN Visiting Research Scientist), Graduate Student Taiki Yoneda (at the time of the research), Graduate Student Taito Imada from the Graduate School of Bioresources at Mie University and RIKEN Team Directors Ichiro Hiratani and Itoshi Nikaido (Professor at Institute of Science Tokyo) has developed a new single-cell analysis technology called scRR-seq (scRepli-RamDA-seq). This method enables high-resolution simultaneous analysis of both genomic DNA and RNA within a single cell. Given it can directly link changes in DNA with changes in gene expression, the technology is expected to help solve problems that were difficult to approach with conventional techniques. The study was published in Nature Communications.

Single-cell analysis technology has advanced significantly, revealing cell-to-cell heterogeneity (the individuality of each cell). Such uniqueness cannot be captured by analyzing cell populations in itself. This has brought innovation to various fields of biology and led to numerous discoveries, including the identification of rare abnormal cells.

However, most existing single-cell analysis methods handle DNA and RNA separately, which has limited our understanding of how these molecules are functionally connected.

The joint research group had previously developed scRepli-seq, high-resolution sequencing analysis of DNA copy number, and RamDA-seq, a highly sensitive RNA sequencing method. This time, they combined these two advanced single-cell analysis methods and designed scRR-seq to enable simultaneous analysis of DNA and RNA from a single cell.

In this new method, magnetic beads containing carboxyl groups are first added to a solution containing DNA and RNA obtained by lysing a single cell. The DNA binds to the beads is separated using a magnetic stand. This allows the RNA to remain in the lysate while the DNA is retained on the beads. Following separation, the DNA is analyzed by scRepli-seq while the RNA is examined using RamDA-seq.

Using the DNA copy number data obtained with this new method, it is possible to estimate what stage of S-phase each cell is in within the cell cycle. Since RNA can be examined simultaneously, changes in gene expression according to S-phase progression can be tracked. This opens the potential for the identification of genes which serve as markers for S-phase progression.

When the researchers examined this using mouse ES cells, they discovered genes whose expression changes according to S-phase progression (S-phase progression markers). Analysis of these genes confirmed clear changes in expression aligning with S-phase progression. When these markers were applied to publicly available single-cell RNA analysis data of mouse ES cells, the S-phase position of each cell could be predicted, and the pattern of S-phase progression was reproduced.

Next, they used this technology to tackle problems which were difficult to approach previously. In the field of DNA replication research, understanding how replication-associated changes in animal cells' DNA copy number affect gene expression has been a long-standing challenge. In bacteria, gene expression positively correlates with DNA copy number, but it was unknown whether the same was true for animal cells with larger and more complex genomes.

Therefore, the group performed analysis using human HAP1 cells, which have only one copy of each chromosome. When focusing on S-phase cells undergoing replication, they found that some genomic regions were already replicated (2 copies) while others remained unreplicated (1 copy). They verified whether expression was higher in cells with 2 copies rather than cells with 1 copy for each gene, and surprisingly, many genes showed no significant increase or decrease in expression after replication.

This suggests that, unlike bacteria, animal cells may possess robust regulatory mechanisms that stabilize gene expression against changes in DNA copy number caused by replication.

There is great potential within this new method to become a next-generation integrated single-cell DNA/RNA analysis technology with broad applications from basic biology to clinical research.

Journal Information
Publication: Nature Communications
Title: scRepli-RamDA-seq: a multi-omics technology enabling the analysis of gene expression dynamics during S-phase
DOI: 10.1038/s41467-025-64688-1