Single-Cell RNA Sequencing of Murine Liver Reveals an Aligned Circadian Clock and Cell-Population-Specific Circadian-Regulated Pathways

The circadian clock is tightly connected to metabolism, which is evident in the various metabolic processes performed by the liver. Perturbation of these processes due to circadian dysregulation leads to liver-specific pathology. The liver is composed of multiple cell populations, each with distinct functions contributing to organ homeostasis, but the individual contributions of these populations to circadian clock function are not yet known. Single-cell RNA sequencing provides the opportunity to understand clock function and oscillating gene expression within an organ system at the individual cell population level, allowing for a better understanding of the crosstalk between the circadian clock and metabolic pathways within the liver. Previously, barriers to achieving this goal included both the complexity of generating single-cell RNA-sequencing time series data and the complexity of data analysis. Herein, we established a protocol that enabled the generation of murine liver cell population time series data, as well as a methodological approach to evaluate the core molecular clock and oscillating gene expression in individual cell populations. Using a combination of the normalized coefficient of variation, clock-correlation, and pseudobulk analyses, we found a robust and aligned circadian clock in each of the hepatic cell populations. We then employed a pseudoreplicate/pseudobulk strategy to identify oscillating gene expression, and when benchmarked against bulk RNA-sequencing data, we demonstrated that many metabolic genes were oscillating in several of the cell populations, including non-hepatocyte clusters. Finally, we identified oscillating genes unique to specific cell populations that play critical roles in liver function. The findings in this study provide a critical foundation for understanding clock function and the contributions of oscillating gene function at the individual cell population level in the liver.