Fig. 4

Transcriptional heterogeneity of single hESCs. a Part of the barcode matrix used for the analysis of single (1) and multiple (2, 4, 8, 16, 32) hESCs maintained by different media (mTeSR™1, KSR-bFGF, and E8) and BJ fibroblasts. Negative controls were wells not receiving sorted cells (0). Prior to sorting, all wells (including negative controls) were pre-filled with 2 μl of RT mixture containing fixed concentrations of four RNA spike-ins. Over 4500 wells representing two biological replicates were analyzed as two libraries and sequenced using Illumina NextSeq for a total of 23.5 million processed paired reads. b Normalized read counts of selected genes plotted against the number of cells sorted per well (n = 858 samples from KSR-bFGF medium are shown). Correlation coefficients (R) between the cell counts and the median of corresponding reads are shown. c Violin plots illustrating the expression of a subset of genes by hESCs and fibroblasts. Samples include single cells and calculated one-cell values of multi-cell wells. Higher B2M expression by fibroblasts was noted [23], while pluripotency and cell cycle genes had notably higher expression in the hESCs. RNA1 represent the spike-ins. d UMAP projection of single hESCs (n = 1550) treated with three media (black dot, mTeSR; orange dot, bFGF; light blue dot, E8), with respect to 11 genes. Expression of some of the genes underlying the distribution is plotted on the right. All results are based on two biological replicates, and plots for the rest of the genes (and conditions) for b and d are shown in Additional file 7: Figure S4