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We infected cells with pCW-Cas9 plasmid (Addgene #50661) and selected cells with puromycin at 2 μg/mL. To improve knockout efficiency, cells were then infected with 2 distinct KIF18A sgRNA plasmids. Each sgRNA sequence was cloned into its own lenti-sgRNA-blast plasmid (Addgene #104993) and these plasmids were co-packaged into lentivirus and used to infect cells, which were then selected with blasticidin (Sigma) at 5μg/mL. The sequences for both KIF18A targeting sgRNAs are available in ref38:
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