To generate U2OS and 293T CATACOMB KO cells, we used two guides (sequence 1: 5′-CACCGCAACCAGGGTCGACCCCAG-3′; sequence 2: 5′-CACCGACAGCAGGTTAAGTGCTAGG-3′); each of them was cloned in pX459 V2.0 (a gift from F. Zhang, Addgene plasmid #62988). Both plasmids were cotransfected in U2OS using Lipofectamine 3000 (Thermo Fisher Scientific) or using calcium phosphate in 293T. Cells were selected with puromycin (2 μg/ml) for 2 days and then switched to medium without antibiotic to avoid plasmid integration. Clones were then obtained by limited dilution single cell cloning. Clones were screened by PCR for CATACOMB locus deletion and then verified by Western blot analysis. To generate the mESC Ezh2ΔSET, we used two guides (sequence 1: 5′-CACCGGGGTCTAGTGATTCCCCTC-3′; sequence 2: 5′-CACCGTTTCACGTAAGTACTCCAG-3′); each of them was cloned in pX459 V2.0. Both plasmids were electoporated in mESCs. Cells were selected with puromycin (2 μg/ml) for 2 days and then switched to medium without antibiotic to avoid plasmid integration. Cells were then plated at low density, and single colonies were picked. Clones were screened by PCR for the Ezh2 SET domain deletion and then verified by Western blot.

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