The workflow to generate and identify knockout cell clones using OutKnocker. (A) Schematic view of the developed workflow to obtain gene-targeted cell clones for subsequent deep sequencing analysis. Forty-eight hours after transfection of a designer nuclease, cells are seeded under limiting dilution conditions and cultured for 2 wk. Grown single-cell clones are picked and duplicated. One duplicate is lysed to perform a locus-specific PCR and a subsequent second PCR is performed to attach barcodes and sequencing adapters. Obtained PCR products are then pooled and subjected to deep sequencing. (B) Schematic view of the alignment and indel calling algorithm used by OutKnocker (see text for details).
