Modification of Seurat v4 for the development of a phase assignment tool able to distinguish between G2 and Mitotic cells

Single cell RNA sequencing (scRNAseq) is a rapidly advancing field which allows for the characterization of the cellular heterogeneity of gene expression profiles within a population. Cell cycle phase is a major contributor to gene expression variance between cells and computational analysis tools have been developed to assign cell cycle phase to scRNAseq datasets. Whilst these tools can be extremely useful, all have the drawback that they classify cells as G1, S or G2/M. Discrete cell phase assignment tool are unable to differentiate between G2 and M and continuous phase assignments tools are unable to identify a region corresponding specifically to mitosis in a pseudo-timeline for continuous assignment along the cell cycle. Bulk RNA sequencing was used to identify differentially expressed genes between mitotic and interphase cells isolated based on phospho-histone H3 expression using fluorescence activated cell sorting. The gene lists were used to develop a Modified Seurat Mitotic Sort (MoSMiS) methodology which can distinguish G2 and M phase cells in single cell RNA sequencing data. The phase assignment tools present in Seurat were modified to allow for cell cycle phase assignment of all stages of the cell cycle identifying a mitotic specific cell population.