Cell culture

Human HeLa (CCL-2) and U2OS (HTB-96) cells were obtained from the American Type Culture Collection. HEK293-EBNA1-6E cells were a kind gift from Yves Durocher (National Research Council Canada). HeLa, U2OS and HEK293-6E cells were propagated in DMEM supplemented with 10% (v/v) FBS and 1% penicillin–streptomycin. HAP1 cells53 were cultured in IMDM (Gibco) supplemented with 10% heat-inactivated FCS (Thermo Fisher Scientific) and penicillin–streptomycin–glutamine solution (Gibco). HeLa cells stably expressing GFP–DNMT1 were described previously11. To generate inducible cell lines expressing WT and mutant GFP–TOPORS proteins, pcDNA5/FRT/TO/GFP/TOPORS plasmids were co-transfected with pOG44 into U2OS Flp-In T-Rex cells (Thermo Fisher Scientific) using Lipofectamine 3000 (Invitrogen). Clones were selected in medium containing hygromycin B (Thermo Fisher Scientific) and blasticidin (InvivoGen) and verified by microscopy. All cell lines were regularly tested negative for mycoplasma infection and were not authenticated.

Plasmid DNA transfections were performed using FuGENE 6 (Promega), Lipofectamine 2000 (Invitrogen) or Lipofectamine 3000 (Invitrogen) according to the manufacturers’ protocols. Cell cycle synchronizations were performed as previously described54, using single treatment with thymidine for 18 h. Unless otherwise indicated, the following doses of chemicals and genotoxic agents were used: 5‐AzadC (10 μM, Sigma‐Aldrich), EdU (20 µM, Sigma‐Aldrich), arsenic trioxide (arsenic; 1 µM, Sigma‐Aldrich), formaldehyde (500 µM, Thermo Fisher Scientific), camptothecin ((CPT); 10 µM; AH Diagnostics); FT-671 (USP7i; 2 µM, MedChemExpress), MG132 (20 μM, Sigma‐Aldrich), ML‐792 (SUMOi; 2 μM, MedKoo Biosciences), MLN‐7243 (Ub-E1i; 5 μM, Active Biochem), NMS-873 (p97i; 5 μM, Sigma‐Aldrich), dTAG-13 (0.25 μM, Tocris Biosciences) and thymidine (2 mM, Sigma‐Aldrich).

Plasmids

Full-length cDNAs encoding human TOPORS WT (codon optimized, Invitrogen GeneArt), *RING (I105A,L140A,K142A; codon optimized) and TOPORS *SIM (V391A,I392A,I394A,V479A,I480A,V481A,V484A,L494A,V495A,L497A,V906A,V907A,I908A,I910A; codon optimized) were PCR amplified and cloned into pcDNA5/FRT/TO/GFP via KpnI and NotI (for generation of stable cell lines) or pcDNA4/TO/Strep–HA via EcoRV and NotI (for protein expression).

siRNAs

siRNA transfections were performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. All siRNAs were used at a final concentration of 20 nM. The following siRNA oligonucleotides were used: non‐targeting control (CTRL): 5′-GGGAUACCUAGACGUUCUA‐3′; RNF4: 5′-GAAUGGACGUCUCAUCGUU-3′; TOPORS(#1): 5′-GUCCUAAGGCCUUCGUAUAAU-3′; TOPORS(#2): 5′-CCCUGCUCCUUCAUACGAA-3′; SUMO1 (5′-GGACAGGAUAGCAGUGAGA-3′); SUMO2/3 (5′-GUCAAUGAGGCAGAUCAGA-3′); UBE2K(#1): 5′-GAAUCAAGCGGGAGUUCAA-3′; UBE2K(#2): 5′-CCUAAGGUCCGGUUUAUCA-3′; UBE2K(#3): 5′-CCAGAAACAUACCCAUUUA-3′ and UBE2K(#4): 5′-GCAAAUCAGUACAAACAAA-3′ (a 1:1:1:1 mix of all four UBE2K siRNAs was used).

Immunoblotting, immunoprecipitation and chromatin fractionation

Immunoblotting was performed as previously described55. To prepare cell extracts, cells were lysed in EBC buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 1 mM EDTA; 0.5% NP40; 1 mM DTT) or MultiDsk lysis buffer (50 mM Tris, pH 8.0; 1 M NaCl; 5 mM EDTA; 1% NP40; 0.1% SDS) supplemented with protease and phosphatase inhibitors on ice for 20 min, and lysates were cleared by centrifugation (21,000g, 20 min). For immunoprecipitation (IP) experiments to analyze protein modification by SUMO and ubiquitin, cells were lysed in denaturing buffer (20 mM Tris, pH 7.5; 50 mM NaCl; 1 mM EDTA; 0.5% NP40; 0.5% SDS; 0.5% sodium deoxycholate; 1 mM DTT) supplemented with protease and phosphatase inhibitors, followed by sonication. For co-IP experiments, cells were lysed in EBC buffer supplemented with protease and phosphatase inhibitors on ice for 20 min. Lysates were then cleared by centrifugation (21,000g, 20 min) and incubated with GFP-Trap or DNMT1-Trap agarose (ChromoTek) overnight at 4 °C. After washing, immobilized proteins were eluted from the beads by boiling in 2× Laemmli sample buffer for 5 min. For two-step IPs, GFP‐tagged DNMT1 was purified on GFP‐Trap agarose (ChromoTek) under denaturing conditions as described above and washed extensively in denaturing buffer. Beads were equilibrated in EBC buffer and incubated with whole-cell lysate prepared in EBC buffer at 4 °C for 4 h. Beads were then washed in EBC buffer, and proteins were eluted by boiling in 2× Laemmli sample buffer for 5 min.

For pulldowns of total cellular ubiquitin conjugates using the MultiDsk affinity reagent45, purified Halo-tagged MultiDsk (15 μg per condition) was pre-incubated with HaloLink resin (Promega) for 1 h at room temperature in binding buffer (100 mM Tris, pH 7.5; 150 mM NaCl; 0.05% IGEPAL). Excess protein was removed by washing with binding buffer supplemented with 1 mg ml−1 BSA. Cells were lysed in MultiDsk buffer supplemented with protease and phosphatase inhibitors on ice for 15 min, followed by sonication. Lysates were then cleared by centrifugation (21,000g, 20 min) and incubated with Halo-MultiDsk resin overnight at 4 °C. After multiple washes of beads, ubiquitylated proteins were eluted by boiling in 2× Laemmli sample buffer for 5 min.

For chromatin fractionation, cells were first lysed in buffer 1a (10 mM Tris, pH 8.0; 10 mM KCl; 1.5 mM MgCl2; 0.34 M sucrose; 10% glycerol; 0.1% Triton X‐100) supplemented with protease and phosphatase inhibitors on ice for 5 min, followed by centrifugation (2,000g, 5 min) to recover the soluble proteins. Pellets were then washed once in buffer 1b (buffer 1a supplemented with 500 mM NaCl) and once in buffer 1a, followed by resuspension in buffer 2 (50 mM Tris, pH 7.5; 150 mM NaCl; 1% NP40; 0.1% SDS; 1 mM MgCl2; 125 U ml−1 benzonase) supplemented with protease and phosphatase inhibitors. Lysates were incubated in a thermomixer (37 °C, 1,000 r.p.m., 15 min), and solubilized chromatin-bound proteins were obtained by centrifugation (21,000g, 10 min).

Antibodies

Antibodies to human proteins used in this study included: actin (1:20,000 dilution (MAB1501, Millipore, RRID: AB_2223041)); DNMT1 (1:1,000, described in ref. 11); FLAG (1:1,000 (A00187, GenScript, RRID: AB_1720813)); GFP (1:5,000, Abcam (ab6556, RRID: AB_305564)); HA (1:1,000 (11867423001, Roche, RRID: AB_390918)); histone H3 (1:20,000, Abcam (ab1791, RRID: AB_302613)); PARP1 (1:1,000, Santa Cruz Biotechnology (sc-8007, RRID: AB_628105)); PIAS1 (1:1,000 (ab77231, Abcam, RRID: AB_1524188)); RNF4 (1:5,000, described in ref. 54); SUMO1 (1:1,000, Thermo Fisher Scientific (33-2400, RRID: AB_2533109)); SUMO2/3 (1:1,000, Abcam (ab3742, RRID: AB_304041); 1:1,000, Abcam (ab81371, RRID: AB_1658424)); TOP1 (1:500, Bethyl (A302-590A, RRID: AB_2034875)); TOPORS (1:250, sheep polyclonal raised against full-length human TOPORS); ubiquitin (1:1,000, Santa Cruz Biotechnology (sc-8017 AC, RRID: AB_2762364); 1:1,000, Millipore (04-263, RRID: AB_612093)); ubiquitin (K48-linked) (1:1,000, Millipore (05-1307, RRID: AB_1587578)); ubiquitin (K63-linked) (1:1,000, (05-1308, RRID: AB_1587580)); UBE2K (1:1,000, Cell Signaling Technology (3847, RRID: AB_2210768)); and USP7 (1:1,000, Bethyl (A300-033A, RRID: AB_203276)).

Immunofluorescence and high‐content image analysis

Cells were pre-extracted on ice in stringent pre-extraction buffer (10 mM Tris-HCl, pH 7.4; 2.5 mM MgCl2; 0.5% NP‐40; 1 mM PMSF) for 8 min and then in ice-cold PBS for 2 min before fixation with 4% formaldehyde for 15 min. If not pre-extracted, cells were subjected to permeabilization with PBS containing 0.2% Triton X‐100 for 5 min before blocking. Coverslips were blocked in 10% BSA and incubated with primary antibodies for 2 h at room temperature, followed by staining with secondary antibodies and DAPI (Alexa Fluor, Life Technologies) for 1 h at room temperature. Coverslips were mounted in Mowiol 4‐88 (Sigma-Aldrich).

Manual image acquisition was performed with a Zeiss LSM 880 laser scanning confocal microscope, using a Plan-Apochromat ×40 1.30 oil DIC M27 objective and ZEN software (Zeiss). Coverslips were prepared as described above, except that they were mounted with VECTASHIELD Mounting Medium (Vector Laboratories) and sealed with nail polish. Raw images were exported as TIFF files and processed using Adobe Photoshop. If adjustments in image contrast and brightness were applied, identical settings were used on all images of a given experiment. For automated image acquisition, images were acquired with an Olympus Ixplore ScanR system equipped with an Olympus IX‐83 wide‐field microscope, a Yokogawa CSU-W1 confocal spinning disk unit, four 50/100-mW laser diodes, UPLSXAPO20× NA 0.80 WD 0.60 mm or UPLSXAPO40×2 NA 0.95 WD 0.18 mm and a Hamamatsu Orca Flash 4 sCMOS camera. Automated and unbiased image analysis was carried out with ScanR analysis software. Data were exported and processed using Spotfire (TIBCO Software).

Live-cell imaging

U2OS cells stably expressing Fucci reporter constructs (mKO2-hCdt1(30-120) and mAG-hGem(1-110))48 were transfected with siRNAs and seeded onto an ibidi dish 16 h before acquisition. Medium was changed to Leibovitz’s L15 medium (Life Technologies) supplemented with 10% FBS before filming. Live-cell imaging was performed on a DeltaVision Elite system using a ×40 oil objective (GE Healthcare). Images were acquired every 10 min or 15 min for 48 h. Three z-stacks of 5 μm were imaged. SoftWork software (GE Healthcare) were used for data analysis.

Proliferation and survival assays

Relative proliferation was measured using an IncuCyte (Sartorius) instrument. Cells (6 × 104) were seeded into 24-well or 96-well plates 24 h after siRNA treatment and imaged every 2 h. Mean confluency was determined from four (Fig. 5e) or nine (Extended Data Fig. 6f,g) images. For survival assays, approximately 400 cells were plated per 60-mm plate and treated with 5-AzadC for 24 h or formaldehyde for 30 min. Cells were then washed with PBS twice, and fresh medium was added. Colonies were fixed and stained after approximately 2 weeks with cell staining solution (0.5% w/v crystal violet, 25% v/v methanol). The number of colonies was quantified using a GelCount (Oxford Optronix) colony counter.

Purification of recombinant proteins

For purification of Strep–HA–TOPORS proteins, HEK293-6E suspension cells were cultured in FreeStyle F17 expression medium (Gibco) supplemented with 4 mM L-glutamine (Gibco), 1% FBS, 0.1% Pluronic F-68 (Gibco) and 50 μg ml−1 G418 (Invivogen) in a 37 °C incubator on a shaker rotating at 140 r.p.m. HEK293-6E cells were transfected with Strep–HA–TOPORS WT, *SIM or *RING expression plasmids using PEI transfection reagent (PolyScience). After 24 h, cells were harvested and snap frozen in liquid nitrogen. The cell pellet was resuspended in lysis buffer (100 mM Tris, pH 8.0; 350 mM NaCl; 0.05% NP-40; protease inhibitors), treated with benzonase and sonicated. The lysate was then cleared by centrifugation at 25,000g at 4 °C for 30 min. The cleared lysate was incubated with Strep-Tactin Superflow resin (IBA Lifesciences) and incubated at 4 °C for 2 h. Beads were washed in a gravity column using wash buffer (100 mM Tris, pH 8.0; 600 mM NaCl; 0.05% NP-40), and TOPORS protein was eluted using elution buffer (100 mM Tris, pH 8.0; 350 mM NaCl; 2.5 mM desthiobiotin). The elute fractions were run on a 4–12% NuPAGE Bis-Tris protein gel (Invitrogen) and stained with Instant Blue Coomassie Protein Stain (Expedeon). Fractions were concentrated on Microcon-30kDa Centrifugal Filters (Millipore), snap frozen in liquid nitrogen and stored at −70 °C. For in vitro ubiquitylation and SUMOylation reactions, 0.35 μg of Strep–HA–TOPORS prep was used; for Strep-Tactin pulldowns, 0.7 μg was used; and for STUbL assays, 2.8 μg was used.

Purification of GST–SUMO proteins was described previously56. Plasmids for recombinant expression of GST–COMP, GST–COMP–SUMO1 and GST–COMP–SUMO2 (ref. 46) were a kind gift from Andrew Sharrocks (University of Manchester). The same procedure of expression and purification was followed as for GST–SUMO proteins, except that Rosetta (DE3) BL21 Escherichia coli was used and proteins were not eluted from the glutathione agarose resins before use in assays.

In vitro ubiquitylation, SUMOylation and deubiquitylation reactions

All in vitro ubiquitylation and SUMOylation reactions were carried out in reaction buffer containing 50 mM Tris, pH 7.5; 150 mM NaCl; 0.1% NP-40; 5 mM MgCl2; 0.5 mM TCEP. Unless otherwise stated, the following final concentrations were used: FLAG ubiquitin (R&D Systems, 20 μM), ATP (Sigma-Aldrich, 3 mM), UBA1 (100 nM), UBE2D1 (0.5 μM), UBE2K (R&D Systems, 0.28 μM), SAE1-UBA2 (R&D Systems, 100 nM), UBE2I (R&D Systems, 0.5 μM), SUMO2 (R&D Systems, 2 μM), poly-SUMO22–8 chains (R&D Systems, 0.3 μg per reaction), RNF4 (47 nM), GST–PIAS1 (Enzo Life Sciences), 4×SUMO2 and Ub–4×SUMO2 (1.8 μM)36. HA–SUMO1 vinyl sulfone (VS) and HA–ubiquitin VS (R&D Systems) were added to all reactions containing purified Strep–HA–TOPORS proteins to inhibit deubiquitinase and SUMO protease activities co-purifying with Strep–HA–TOPORS.

For GST–SUMO and GST–COMP–SUMO ubiquitylation assays, GST proteins immobilized on glutathione agarose were incubated at 22 °C with agitation for 1 h with Strep–HA–TOPORS (0.6 µM), His6-UBE1 (0.1 µM), UBE2D1 (1 µM) and 40 µM ubiquitin (10% labeled with 5-IAF) in reaction buffer supplemented with 2 mM ATP. Beads were washed twice with reaction buffer and transferred to a new tube, and bound proteins were eluted with 30 µl of 1× Laemmli sample buffer. Fluorescein-ubiquitin species on gel were visualized by laser scanning (Typhoon, Cytiva Life Sciences) using the Cy2 setting before staining with Coomassie blue. Data for ubiquitin-modified GST substrates from triplicate reactions were quantified by densitometry (ImageJ) from non-saturated negative scans.

For in vitro deubiquitylation reactions with recombinant USP7, GFP–TOPORS expressed in U2OS cells was immunoprecipitated using GFP-Trap agarose under denaturing condition as above, followed by extensive washing. Beads containing bound GFP–TOPORS were equilibrated in deubiquitylation buffer (50 mM Tris, pH 7.5; 150 mM NaCl; 5 mM DTT) and incubated with 0.5 µg of recombinant USP7 protein (Ubiquigent) with shaking at 30 °C for 30 min. Beads were washed, eluted by boiling in 2× Laemmli sample buffer for 5 min and analyzed by immunoblotting.

SUMO-binding assays

Human recombinant SUMO1 or SUMO2 coupled to agarose at 0.5 mg of protein per milliliter of settled resin (Enzo Life Sciences) was incubated with whole-cell extracts from U2OS cells or recombinant Strep–HA–TOPORS *RING protein in EBC buffer overnight with constant agitation at 4 °C. After multiple washes, bound proteins were eluted in 2× Laemmli sample buffer and analyzed by immunoblotting. To assay for TOPORS binding to poly-SUMO2 chains, purified Strep–HA–TOPORS proteins (0.7 μg per reaction) and recombinant poly-SUMO22–8 chains (R&D Systems, 0.3 mg per reaction) were incubated with Strep-Tactin Superflow resin (IBA Lifesciences) and incubated at 4 °C for 2 h in EBC buffer. The beads were then washed in EBC buffer, and bound proteins were eluted in 2× Laemmli sample buffer and analyzed by immunoblotting.

Flow cytometry

Cells were collected by trypsinization, fixed in 4% paraformaldehyde in PBS for 15 min and permeabilized in 0.2% Triton X-100, 2% FBS in PBS for 20 min at room temperature. Permeabilized cells were washed in FACS buffer (PBS + 10% FBS) and incubated with sheep polyclonal anti-DNMT1 antibody15 diluted in FACS buffer for 90 min at room temperature. After two washes in FACS buffer, cells were incubated with anti-sheep Alexa Fluor 488 (Invitrogen) diluted in FACS buffer for 1 h at room temperature. For EdU co-staining, washed cells were subsequently stained using the Click-iT Plus EdU Alexa Fluor 647 Kit (Invitrogen) according to the manufacturer’s instructions. After two additional washes in FACS buffer, cells were resuspended in FACS buffer containing 1 µg ml−1 DAPI (Thermo Fisher Scientific), strained to a single-cell solution (40-µm filter) and analyzed using an LSRFortessa flow cytometer (BD Biosciences). Analysis was performed and plots were generated using FlowJo software (version 10.8.1).

Mutagenesis of HAP1 cells

Gene-trap mutagenesis of HAP1 cell lines was carried out as described57. In brief, a BFP-containing variant of gene-trap retrovirus was generated in low-passage HEK293T cells by co-transfection of gene-trap vector, retroviral packaging plasmids Gag-pol and VsVg and pAdvantage (Promega). Media containing retrovirus were collected 48 h and 72 h after transfection and concentrated using centrifugation filters (Amicon), and the pooled, concentrated retrovirus was used to transduce 40 × 106 HAP1 cells supplemented with protamine sulfate (8 µg ml−1) for 24 h. After recovery, mutagenized HAP1 cells were expanded and used for genetic screens.

FACS-based screens for DNMT1 abundance

To identify regulators of 5-AzadC-induced DNMT1 processing, mutagenized HAP1 WT cells were expanded to 3 × 109 cells per screen. Two screens were performed: a mock DNMT1 screen where cells were labeled for 2 h with 10 µM EdU and a perturbation DNMT1 screen with co-treatment of 10 µM 5-AzadC and EdU. Treated cells were then trypsinized, fixed, permeabilized and stained for DNMT1 and EdU as described in the ‘Flow cytometry’ subsection above. Cells were sorted on a BD FACSAria Fusion cell sorter gating for haploid EdU-incorporating cells, and the cells with the 5% highest and lowest DNMT1 signal, respectively, were collected. Genomic DNA was extracted from sorted cells (1.3 × 107 cells per channel) using the QIAamp DNA Mini Kit (Qiagen). Gene-trap insertion site recovery and sequencing libraries were generated as previously described58. Amplified libraries were sequenced on a HiSeq 2500 (Illumina) with a read length of 65 bp (single-end read). Sequencing reads from each sample (low and high) were aligned to the human genome (hg38) allowing one mismatch and assigned to non-overlapping protein-coding gene regions (RefSeq). The number of unique gene-trap insertions in the sense direction of each gene was normalized to the total number of sense insertions of each sorted population. The mutational index (MI) was calculated for each gene by comparing the number of unique, normalized sense integrations of the high population to that of the low population using a two-sided Fisher’s exact test (false discovery rate (FDR)-corrected P < 0.05). Every gene was then plotted in fishtail scatterplots comparing the combined number of unique insertions identified in the two populations of a given gene (log10, x axis) to its MI (log2, y axis). To identify genes selectively affecting DNMT1 abundance in response to 5-AzadC, but not in the mock screen, a comparative filtering of genes was performed. Significant positive and negative regulators that scored as such in both screens (except for the antigen target DNMT1) were removed from the 5-AzadC screen to highlight DNMT1 DPC-specific regulators and generate Fig. 1b. Significant regulators for each individual screen can be found in Supplementary Data 1. Fishtail scatterplots were generated using GraphPad Prism software.

Fitness-based screens to identify synthetic lethal interactions

Haploid genetic fitness screens were carried out as described in ref. 47. In brief, mutagenized HAP1 cell lines (minimum coverage of 2.5 × 108 cells per screen) were passaged for 10 d, trypsinized and fixed using Fixation Buffer I (BD Biosciences) for 10 min at 37 °C. For RNF4–dTAG cells, cells were passaged in the presence of 0.25 µM dTAG-13 to induce loss of RNF4. Cells were then permeabilized with Perm Buffer III (BD Biosciences) and stained in FACS buffer containing 2.5 µg ml−1 DAPI (Thermo Fisher Scientific). After washing in FACS buffer, cells were strained to a single-cell solution (40-µm filter), and a minimum of 3 × 107 haploid cells (based on DAPI content) were isolated using a BD FACSAria Fusion cell sorter. Isolation of genomic DNA, library preparation and insertion site mapping were done as described for the FACS-based screens above. Analysis of gene-trap insertion orientation bias (sense ratio) was performed as previously described47, and the analysis pipeline can be found on GitHub (https://github.com/BrummelkampResearch/phenosaurus). For each replicate experiment, a two-sided binomial test was calculated, which gives a P value for each gene. These P values were then corrected for FDR using the Benjamini–Hochberg procedure, and the least significant P value among the individual replicates of a genotype was used to determine if a gene was considered significant. Every replicate corresponds to an independent clonal cell line of the respective genotype. Four independent cultured WT control datasets published in ref. 47 were used as control (available at Sequence Read Archive SRP058962, accession numbers SRX1045464, SRX1045465, SRX1045466 and SRX1045467). To identify genes that affect cell viability selectively in TOPORS-deficient and RNF4-deficient cell lines, the number of disruptive sense integrations and non-disruptive antisense integrations for each gene was compared to that in the four control datasets using a two-sided Fisher’s exact test. Genes with a significant orientation bias in screen replicates of TOPORS-deficient and RNF4-deficient cells, respectively, in addition to a significantly altered sense ratio (P < 0.05, odds ratio < 0.8) in relation to the control datasets, were considered as hits.

Generation of HAP1 cell lines

To generate clonal TOPORS-KO cell lines, parental HAP1 cells were co-transfected with a blasticidin resistance cassette and the CRISPR–Cas9 vector pX330 containing sgRNA sequences targeting TOPORS (sgTOPORS1 (5′-AACAGTACTCCACTATCCGG-3′) and sgTOPORS2 (5′-GGTAGCGAAATCGTCGATCA-3′)). Cells were then briefly selected (48 h) during clonal outgrowth, and gene status was monitored using Sanger sequencing of genomic DNA and immunoblot analysis. C-terminal degron tagging of endogenous RNF4 was carried out by a generic CRISPR–Cas9 strategy as previously described59 but using a modified pTIA donor vector containing HA-tagged FKBP12(F36V) (dTAG) and a P2A sequence followed by a blasticidin cassette for integration selection (pTIA dTAG–HA P2A Blast). To generate clonal cell lines, parental HAP1 cells were co-transfected (1:1) with pTIA dTAG donor vector and a CRISPR–Cas9 vector containing an sgRNA targeting the last exon (exon 8) of RNF4 (pX330 sgRNF4 (5′-TACTTCATATATAAATGGGG-3′)) and subjected to blasticidin selection during clonal outgrowth. HAP1 RNF4–dTAG clones were validated by Sanger sequencing of the genomic locus and immunoblot analysis. The two RNF4–dTAG clones used contained an in-frame insertion of the dTAG donor vector at the C-terminus of RNF4, after amino acid His186 (hg38 genome coordinate chr4:2,513,804).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.