Single-cell multiplexed qPCR data was analyzed and displayed using custom R scripts12 . qPCR experiments were performed for E16.5 (2 biological replicates), E18.5 (2 biological replicates) distal lung epithelial cells and for adult AT2 cells (1 replicate). The limit of detection of multiplexed qPCR values was determined as 22 threshold cycles (Ct) by a calibration experiment with 16-fold serial dilutions of total lung cDNA and 6 replicates for each concentration. Genes that were not expressed were given a value higher than the limit of detection and the limit of detection was subtracted from all Ct values to transform Ct values to log2 expression values (log2Ex = CtLoD – Ct, CtLoD = 22). Cells not expressing either of two housekeeping genes Actb and Gapdh, or expressing them below three standard deviations below the mean, were scored as unhealthy and removed from the analysis. After applying this filter, 74 single cells remained for two experiments at E18.5, 33 cells for two experiments at E16.5 and 48 cells for the experiment with adult AT2 cells. In all experiments, cells were isolated from pooled lungs from one litter (5-9 lungs). To combine experiments from different chips for the same embryonic time point, the expression value of each gene for a given cell was normalized to the median gene expression value of that cell. Normalized gene expression values were further scaled gene-by-gene by mean-centering and dividing by the standard deviation of expressing cells. PCA and hierarchical clustering using Euclidean distance metric were performed in R for all cells using 10 canonical marker genes for bronchiolar and alveolar cells (Abca3, Sftpb, Muc1, Sftpc, Lyz2, Aqp5, Pdpn, Ager, Foxj1, Scgb1a1).