Figure 3
Cellular barcoding can reliably detect clonal status of cell lines during single-cell cloning (a) Stable barcoded expression pools were single cell cloned by limited dilution in 384 well plates. Monoclonality was assessed by fluorescent imaging directly after seeding at d0 and barcodes were detected via amplicon deep sequencing at day 18. Wells were grouped based on the initial image based classification in either wells with 1 cell or > 1 cell. (b) Bar graphs depicting average number of barcodes detected by the barcoding method as compared to an automated image analysis method. Samples are grouped according to initial image classification to wells containing only 1 cell and > 1 cell, n = 96. Error bars indicate SD. (c) Fluorescence imaging at d0 directly after seeding of barcoded stable pools in 384 well plates. This image was used for initial classification of wells. (d) Bright-field imaging at day 2 after single-cell cloning (left panel) and magnified view on the cell colonies (right panel). Cell colonies with visible division are marked with a rectangle, cells without visible division are marked by an arrow. Size bar indicates 200 µm. (e) The number of barcodes were detected via amplicon deep sequencing and unique top 100 barcodes are plotted. Dashed line indicates the minimum read count cutoff to discriminate erroneous barcodes from genuine barcodes using an unbiased knee point detection algorithm. (c) Initial fluorescent imaging directly after seeding cells into 384-well plates during single-cell cloning. Cells are marked by an arrow (d).
