Monocle orders individual cells by progress through differentiation. A) An overview of the Monocle algorithm. B) Cell expression profiles (points) in a two-dimensional independent component space. Lines connecting points represent edges of the MST constructed by Monocle. Solid black line indicates the main diameter path of the MST and provides the backbone of Monocle's “pseudo-time” ordering of the cells. C) Expression levels for differentially expressed genes identified by Monocle (rows), with cells (columns) shown in pseudo-time order. Fibroblasts are excluded. D) Bar plot showing the proportion of MEF2C and MYH2 expressing cells measured by immunofluorescence at the time of collection (upper panel), RNA-Seq at the time of collection (middle panel) or RNA-Seq at pseudo-time (lower panel). MEF2C was considered detectably expressed at or above 100 FPKM, and MYH2 at 1 FPKM. MEF2C exhibits a bimodal pattern of expression across the cells (not shown), and a threshold of 100 FPKM separates the modes. E) Expression levels of key regulators of muscle differentiation, ordered by time collected. (Cyclin-dependent kinase 1, CDK1; Inhibitor of DNA binding 1, ID1; Myogenin, MYOG) F) Regulators from panel D, ordered by Monocle in pseudo-time.