Generation of cell lines with AID-tagged proteins

HeLa (cervical carcinoma) used in this study was a clone expressing the tTA tetracycline transactivator (64 (link)). H1299 cells were obtained from American Type Culture Collection. Cell lines ^AIDCDK1^KO (26 (link)) and ^AIDCDK2^KO (25 (link)) were generated as previously described. ^mAIDCyclin B1^KO was a HeLa cell line expressing cyclin B1-mAID without endogenous cyclin B1 (Adrijana Crncec and RYCP, manuscript in preparation).
^AIDCyclin A^KO cells from HeLa were generated by retroviral infection (25 (link)) using the construct AID-cyclin A in pRevTRE-AID/Hyg, followed by transfection of cyclin A CRISPR-Cas9 in pX330. ^AIDCyclin A^KO cells from H1299 were generated by transfecting H1299 cells with AID-cyclin A in pUHD-SB-AID/Hyg, pSBbi-TIR1-tTA/Pur (26 (link)), cyclin A CRISPR-Cas9 in pX330, and Sleeping Beauty transposase (pCMV(CAT)T7-SB100; a gift from Zsuzsanna Izsvak; Addgene, #34879) before selecting with hygromycin and puromycin for 2 weeks. ^AIDCyclin A^KO cells lacking CDK2 were generated by cotransfecting CDK2 CRISPR-Cas9 in pX330 and a plasmid expressing blasticidin-resistant gene (a gift from Tim Hunt, Cancer Research UK) into ^AIDCyclin A^KO HeLa cells. After enriching the transfected cells with blasticidin selection for 36 h, the cells were recovered in blasticidin-free medium for 48 h. In all the above cell lines, single cell-derived colonies were obtained by limiting dilution in 96-well plates.