Novel Single-Cell Multiomics Approach to Analyze Replication Timing and Gene Expression in Mouse Preimplantation Embryos

Analysis of a cell’s replication timing (RT) provides insight into how genes replicate, early or late, during the S-phase of the cell cycle. RT is cell-type specific, inheritable, and has been correlated to gene expression in normal and diseased states. However, most studies have been limited to somatic cells. Very little is known about RT in early mouse embryos, and how it correlates with the start of transcription during zygote gene activation (ZGA), at the 2-cell stage. In this study, we used a novel in-house single-cell multiomics approach to simultaneously analyze RT and gene expression in individual cells of the mouse 1-cell, 2-cell, and 4-cell embryos. We demonstrated that RT was established at the 1-cell stage prior to ZGA. Surprisingly, we observed RT and gene expression correlation trends that were different in early totipotent embryos, compared to previously published studies in somatic cells. Late replicating regions correlated with higher gene expression and open chromatin in the early developing embryos. Lastly, we performed an integrated pseudo time trajectory analysis combining RT and gene expression information per cell. Mouse zygotes at E0.5 were extracted from super ovulated C57/Bl6 mice. Zygotes were cultured in vitro until 2-cell and 4-cell stages. Zygotes/1-cell embryos were directly transferred into RLT buffer in 96-well plates with 1 zygote/ well. Single cells from 2-cell embryos were extracted 26-28 hours after zygote extraction. Single cells from 4-cell embryos were extracted 38-40 hours after zygote extraction. For 2-cell and 4-cell stages, first the zona pellucida was removed using the Tyrode Acid Solution, then the embryos were incubated in TrypLE: Accutase (1:3) solution for ~10 minutes until the embryos dissociated into single cells. Single cells were then mouth pipetted into individual wells of a 96-well plate containing RLT buffer. The wells were spun down and stored at -20C until further processing.