Pseudo‐time trajectory analysis

Single‐cell trajectories were constructed using a matrix of cells and their gene expression profiles by Monocle (Version3.0). Once the cells were ordered, we were able to visualize the trajectory in the reduced dimensional space. The trajectory was visualized in a tree‐like structure, including tips and branches. Differential Expression Analysis in Monocle was used to identify genes that were differentially expressed between groups of cells and to assess the statistical significance of those findings. We identified key genes related to the development and differentiation process, and grouped genes with similar trends in expression, with the reasoning that such groups might share common biological functions and regulators (Qiu et al., 2017; Trapnell et al., 2014).