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 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 different cell populations each with distinct functions contributing to organ homeostasis, but individual cell population contributions to circadian clock function is 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 that would allow for better understanding of the crosstalk between the circadian clock and metabolic pathways within the liver. In the past, barriers to achieving this goal included complexity associated with generating single-cell RNA sequencing time series data as well as the complexity of data analysis. Here, 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 normalized coefficient of variation, clock-correlation and aggregate pseudobulk, we found a robust and aligned circadian clock in each of the cell populations. We then employed a pseudoreplicate / pseudobulk strategy to identify oscillating gene expression and 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 lay an important foundation for understanding clock function and contributions of oscillating gene function at the individual cell population level in liver.