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. 2020 Jun 5;16(6):e9442. doi: 10.15252/msb.20209442

Figure EV2. Visual Cell Sorting for pooled, image‐based genetic screening.

Figure EV2

  1. Image analysis pipeline to calculate nucleus‐to‐cytoplasm (NC) ratio. Nuclei were segmented using the H3‐Dendra2 signal. Cytoplasmic masks were created by dilating and then removing the nuclear mask. Mean miRFP intensity was measured within each mask, and the nucleus‐to‐cytoplasm (N:C) ratio calculated.
  2. After selective photoactivation on the microscope based on N:C ratio, cells were subject to fluorescence‐activated cell sorting and sorted according to their nuclear localization phenotype. Two days after sorting, cells from each sort bin were re‐imaged in the miRFP channel and the nucleus‐to‐cytoplasm ratio reassessed (n = ˜1,500 per photoactivation bin). Center line, median; box limits, 1st and 3rd quartiles; whiskers, 1.5× interquartile range; points, outliers. R1T2, recombination replicate 1, Visual Cell Sorting technical replicate 2.
  3. Representative images of sorted cells.
  4. Correlation plots of normalized scores calculated for each replicate. r 2, square of Pearson's correlation coefficient.