Cell lines
All cell lines are listed in Supplementary Table 2 and were cultured at 37 °C with 5% CO 2 in DMEM GlutaMAX (Gibco), supplemented with 10% fetal calf serum (Avantor, VWR; Supplementary Table 3) and penicillin–streptomycin (Sigma; Supplementary Table 3).
Compounds and materials
All compounds and instruments are listed in Supplementary Table 3.
Generation of knockout cells
Cells were transfected with Cas9-2A-GFP (Addgene, 48138) containing a guide RNA targeting the gene of interest (sgRNAs are listed in Supplementary Table 4 and plasmids in Supplementary Table 5) using Lipofectamine 2000 (Life Technologies, 11668027). Cells were sorted by fluorescence-activated cell sorting on BFP and GFP and were plated at a low density, after which individual clones were isolated. Isolated knockout clones were verified by Sanger sequencing and/or western blot analysis (primers and antibodies are listed in Supplementary Tables 6 and 7, respectively).
PGK plasmids with GFP-tagged protein
The CFAP20 gene was amplified from cDNA by PCR and inserted in PGK-EGFP-C1-IRES-PURO, thereby tagging CFAP20 at its N terminus with GFP (primers and plasmids are listed in Supplementary Tables 5 and 6). The CFAP20R100C mutant was generated using site-directed mutagenesis PCR. The CCNC gene was amplified from the CMV-EGFP-CCNC plasmid and inserted in pLenti-PGK-GFP-puro, thereby tagging CCNC at its N terminus with GFP. The CCNCD182A mutant was generated using site-directed mutagenesis PCR. A region spanning the CMV promoter was amplified by PCR and used to replace the PGK promoter in pLenti-PGK-GFP-puro. A fragment encoding RNaseH1 from plasmid pFRT-TO-EGFP-RNaseH1 was amplified by PCR and inserted in pLenti-CMV-GFP-puro. All sequences and plasmids were verified by Sanger sequencing.
Generation of stable cell lines
Cells were transfected with Lipofectamine 2000 (Life Technologies, 11668027) or polyethyleneimine reagent (Brunschwig Chemie, 23966-2) according to the manufacturer’s instructions. All plasmids are listed in Supplementary Table 5. Lentiviral particles were produced by co-transfecting pLenti plasmids with pMDLg-pRRE, pRSV-REV and pCMV-VSVG in a 2:1:1:4 ratio in HEK293T cells by using polyethylenimine reagent. After production, lentivirus was filtered through a 0.44-µm filter and added to RPE1 cells in a complete DMEM medium supplemented with 4 µg ml−1 polybrene and 10 mM HEPES. After overnight incubation, the medium was removed, and fresh medium was added. The expression of GFP was verified three days after lentiviral transduction.
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