In preparation for cell sorting, we cultivated cells harboring each library in liquid LB medium enriched with chloramphenicol. Subsequently, we diluted 20 µL of the cultures in 1 mL of cold filtered Dulbecco’s PBS (Sigma-Aldrich #D8537) in 15 mL FACS tubes. We performed FACS-sorting on a FACS Aria III flow cytometer (BD Biosciences, San Jose, CA) using a 70 µm nozzle. We utilized a 488 nm laser for detecting forward scatter (FSC) and side scatter (SSC) with a 488 nm/10 nm band-pass filter. This calculation aims to ensure that despite a substantial reduction in cell numbers post-sorting, each unique sequence remains adequately represented in the surviving cell population.