Cell-to-cell transcriptional variability in otherwise homogeneous cell populations plays an important role in tissue function and development. Single-cell RNA sequencing can characterize this variability in a transcriptome-wide manner. However, technical variation and the confounding between variability and mean expression estimates hinder meaningful comparison of expression variability between cell populations. To address this problem, we introduce an analysis approach that extends the BASiCS statistical framework to derive a residual measure of variability that is not confounded by mean expression. This includes a robust procedure for quantifying technical noise in experiments where technical spike-in molecules are not available. We illustrate how our method provides biological insight into the dynamics of cell-to-cell expression variability, highlighting a synchronization of biosynthetic machinery components in immune cells upon activation. In contrast to the uniform up-regulation of the biosynthetic machinery, CD4+ T cells show heterogeneous up-regulation of immune-related and lineage-defining genes during activation and differentiation.NE was funded by the European Molecular Biology Laboratory (EMBL) international PhD programme. ACR was funded by the MRC Skills Development Fellowship (MR/P014178/1). SR was funded by MRC grant MC_UP_0801/1. JCM was funded by core support of Cancer Research UK and EMBL. CAV was funded by The Alan Turing Institute, EPSRC grant EP/N510129/1
