Amplification of the plasmid library

The human Brunello CRISPR knockout pooled library was a gift from D. Root and J. Doench (Addgene, 73178)⁵³. In brief, the amplification of the plasmid DNA library was achieved by retransforming 100 µl ElectroMAX Stbl4 electrocompetent cells (Invitrogen, Thermo Fisher Scientific) with 400 ng of the plasmid library in four separate repetitions. For each repetition, the transformation was performed using 25 µl of cells and 100 ng of the library DNA with an ice-cold electroporation cuvette (0.1 cm gap size, Bio-Rad). Cells were shocked at 1.8 kV (preset settings, Ec1; Gene Pulser Xcell Electroporation Systems, Bio-Rad) and immediately mixed with 980 µl prewarmed (30 °C) SOC-medium (NEB). The electroporated cells in SOC-medium (4 ml in total) were combined in a ventilated falcon tube, and 6 ml of additional SOC-medium was added. Next, the 10 ml culture was shaken at 30 °C for 1 h, plated on 12 × 15 cm LB agar plates containing 100 µg ml⁻¹ carbenicillin and incubated overnight for 16 h at 30 °C. The bacteria were next scraped from the plate using a cell scraper after adding 10 ml ice-cold LB medium per plate. The cell suspension was centrifuged (4 × 30 ml) at 4,000 relative centrifugal force (r.c.f.) for 10 min. The plasmid DNA library was extracted from the pellet using the Plasmid Maxi Kit (QIAGEN).