A single-cell multiomics approach for simultaneous analysis of replication timing and gene expression

Replication timing (RT) allows us to analyze temporal patterns of genome-wide replication, that is, if genes replicate early or late during the S-phase of the cell cycle. RT has been linked to gene expression in normal and diseased states such as cancer. However, studies done to date were on bulk cell populations that required tens of thousands of cells for RT analysis. Here, we developed an affordable novel single cell (sc)-multiomics approach to simultaneously analyze RT and gene expression from cells or nuclei. We used this approach to generate sc-RT profiles and sc-gene expression data from the well-established human liver cancer cell line, HepG2. We demonstrated that as few as 17 mid S-phase cells were sufficient to produce cell-type specific pseudo bulk RT profiles, that had a high correlation to previously published HepG2 bulk RT profiles. The sc-RT profiles allowed us to visualize how individual cells progressed through genome replication. We were also able to demonstrate high-resolution correlations between RT and gene expression within each individual cell, which to our knowledge, has not been done before. We observed trends conserved between individual cells, as well as cell-to-cell variations, which has not been possible so far with the bulk RT studies. gDNA and mRNA were simultaneoulsy extracted from single HepG2 nuclei. gDNA was processed using the sc-Repiseq and Kronos computational pipelines for Replication Timing (RT). mRNA was processed for gene expression analysis. RT and gene expression were analysed per cell and compared to bulk datasets. Correlations between RT and gene expression were drawn within each cell and across the HepG2 population.