Skip to main content
. 2019 May 13;116(22):10842–10851. doi: 10.1073/pnas.1903808116

Fig. 2.

Fig. 2.

Evaluation of the barcode misidentification rate using cells with known phenotype-barcode correspondence. (A) Constructs used to evaluate barcode detection accuracy. The reporter gene luciferase-mCherry is tagged with either a HA tag or a Myc tag to define two phenotypes, as well as a nuclear localization signal to concentrate HA and Myc signals in the nucleus to facilitate detection. The barcodes are placed at the 3′ UTR of the reporter gene, and the correspondence between the barcodes and the HA or Myc tag is determined by sequencing. The reporter gene is driven by a CMV promoter. (B) Images showing HA and Myc immunostaining signals in two different channels. The nuclei with strong HA signals have weak Myc signals and vice versa. The cell boundaries are labeled in green. The nucleus boundaries of HA expression cells and Myc-expressing cells are labeled in red and blue, respectively. (Scale bars: 50 µm.) (C) Scatterplot of the HA and Myc immunostaining intensities of individual cells. Cells assigned to the HA or Myc library based on imaging-based barcode determination are shown in red or blue, respectively. Cells classified as positive on HA or Myc immunostaining (SI Appendix, SI Materials and Methods) are denoted by triangles or circles, respectively. Only 10 out of 1,105 cells with HA-corresponding barcodes are observed as positive in Myc immunostaining, and only 9 out of 1,034 cells with Myc-corresponding barcodes are observed as positive in HA immunostaining, indicating a low barcode misidentification rate. A small fraction of cells (197 out of 2,336 cells) have both HA and Myc immunostaining signals below a threshold value or both HA and Myc immunostaining signals above a threshold value and thus cannot be unambiguously identified as HA-positive or Myc-positive (SI Appendix, SI Materials and Methods). These cells are excluded from analysis. (D) Histogram of the ratio of HA intensity over Myc intensity for individual cells that were decoded to contain HA tag reporter (shown in red) or Myc tag reporter (shown in blue) by barcode imaging.