Plasmid constructs and primers

ES-FUCCI (plasmid # 62451; Sladitschek and Neveu, 2015) and DHB-mVenus (plasmid #136461; Spencer et al., 2013) were purchased from Addgene. The p27 shRNA clone TRCN0000287390 in pLKO1 was obtained from the Sigma MISSION shRNA library. p27 knockdown was tested by qPCR and compared to cells transduced with a scrambled non-targeting control shRNA (Figure 1—source data 1).

The sgRNAs used in this study are listed in the Key Resources Table. sgRNAs were cloned into lentiCRISPR v2 vector at the BsmBI restriction site using Zhang lab protocol (Shalem et al., 2014; Sanjana et al., 2014). p100 sgRNAs were cloned into a modified pLVTHM GFP vector between ClaI and MluI sites or into lentiCRISPR v2 vector.

Deletion of TRAF3 binding motif (amino acids 78–84) from wild-type NIK cDNA was performed by site-directed mutagenesis. NIK-ΔT3 cDNA was cloned into the pWPI mScarlet vector between BamHI and AscI sites.