Cell culture
MDA-MB-453, BT-474, JSC-1 and BC-1 cell lines were acquired from the cryopreserved aliquots of cell lines sourced previously from collaborators or public repositories and extensively characterized as part of the Genomics of Drug Sensitivity in Cancer (GDSC)51,52 and COSMIC Cell Line projects4,53. Bulk cell lines were genotyped by single-nucleotide polymorphism (SNP) and short tandem repeat profiling, as part of the COSMIC Cell Line Project ( and individual clones obtained here were genotyped (Fluidigm) to confirm their accurate identities. MCF10A cells were from M. Jasin’s laboratory (MSKCC). HT-1376 cells were from B. Faltas’s laboratory (Weill Cornell). HEK293FT cells were from T. de Lange’s laboratory (Rockefeller). All cell lines were mycoplasma negative (Mycoalert Detection Kit; Lonza). MDA-MB-453 cells were grown in DMEM:F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. BC-1, BT-474 and JSC-1 cells were grown in RPMI medium supplemented with 10% FBS, 1% penicillin–streptomycin, 1% sodium pyruvate and 1% glucose. HT-1376 cells and HEK293FT cells were grown in DMEM HG medium supplemented with 10% FBS and 1% penicillin–streptomycin. MCF10A cells were cultured in 1:1 mixture of F12:DMEM medium supplemented with 5% horse serum (Thermo Fisher Scientific), 20 ng ml−1 human EGF (Sigma-Aldrich), 0.5 mg ml−1 hydrocortisone (Sigma-Aldrich), 100 ng ml−1 cholera toxin (Sigma-Aldrich) and 10 μg ml−1 recombinant human insulin (Sigma-Aldrich). Unless otherwise noted, all media and supplements were supplied by the MSKCC Media Preparation core facility.
Generation of knockout cell lines
Cells (106) were electroporated using the Lonza 4D-Nucleofector X Unit (MDA-MB-453) or Lonza Nucleofector 2b Device (BT-474, BC-1, JSC-1, HT-1376) using programs DK-100 (MDA-MB-453), X-001 (BT-474, HT-1376) or T-001 (BC-1, JSC-1) in buffer SF + 18% supplement (MDA-MB-453) or 80% solution 1 (125 mM Na2HPO4•7H2O, 12.5 mM KCl, acetic acid to pH 7.75) and 20% solution 2 (55 mM MgCl2) (BT-474, BC-1, JSC-1, HT-1376) and 9 µg (UNG, SMUG1, REV1) or 10 µg (APOBEC3A, APOBEC3B) of pU6-sgRNA_CBh-Cas9-T2A-mCherry plasmid DNA (Supplementary Table 5). mCherry-positive cells were single-cell sorted or bulk sorted and subcloned by limited dilution into 96-well plates by FACS using the FACSAria system (BD Biosciences).
Knockout screening and validation by PCR
CRISPR knockout clone screening
Genomic DNA was isolated using the Genomic DNA Isolation Kit (Zymo Research; ZD3025). Purified genomic DNA for CRISPR–Cas9 knockout screens was amplified using Touchdown PCR. Each PCR reaction comprised 7.4 μl double-distilled H2O, 1.25 μl 10× PCR buffer (166 mM NH4SO4, 670 mM Tris base pH 8.8, 67 mM MgCl2, 100 mM β-mercaptoethanol), 1.5 μl 10 mM dNTPs, 0.75 μl DMSO, 0.25 μl forward and reverse primers (10 μM each), 0.1 μl Platinum Taq DNA Polymerase (Invitrogen; 10966083) and 1 μl genomic DNA. A list of primer sequences is provided in Supplementary Table 5.
PCR for Sanger sequencing
PCR reactions for Sanger Sequencing were performed using the Invitrogen Platinum Taq DNA Polymerase (Invitrogen, 10966083) protocol. Genomic DNA (25 ng) was used for each reaction. A list of the primer sequences is provided in Supplementary Table 5. DNA from PCR reactions was purified from agarose gels using the Invitrogen PureLink Quick Gel Extraction Kit (Invitrogen, K210012). Gel-purified DNA was cloned using the TOPO TA Cloning Kit for Sequencing (Invitrogen; 450030) and colonies were selected for sequencing (Genewiz).
Lentiviral transduction
Lentiviral plasmids for APOBEC3A, APOBEC3B and control knockdown were provided by S. Roberts’ laboratory24. For UNG–GFP lentiviral transduction, UNG2 open reading frames were amplified from a BT-474 cDNA library using the Phusion High-Fidelity polymerase (Thermo Fisher Scientific) and Gibson (NEB) assembled into pLenti-CMV-GFP BlastR (Addgene). The constructs were transfected into HEK293FT cells together with psPAX2 and pMD2.G (Addgene) using calcium phosphate precipitation. Supernatants containing lentivirus were filtered and supplemented with 4 μg ml−1 polybrene. Successfully transduced BC-1 cells were selected by FACS and clones isolated by limiting dilution. For shRNA knockdown, after transduction, cells were selected with hygromycin B.
RNA isolation and quantitative PCR
RNA was isolated using the Quick-RNA Miniprep Kit (Zymo Research; R1054). RNA was quantified and converted to cDNA using the SuperScript IV First-Strand Synthesis System (Invitrogen; 18091050). cDNA synthesis reactions were performed using 2 μl of 50 ng μl−1 random hexamers, 2 μl of 10 mM dNTPs, 4 μg RNA and DEPC-treated water to a volume of 26 μl. The mixture was heated at 65 °C for 5 min, then cooled on ice for 5 min. Primers, probes and cycling conditions were adopted from published methods54. A list of the primer sequences is provided in Supplementary Table 5.
Immunoblotting
Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, Pierce Protease Inhibitor Tablet, EDTA free) or sample buffer (62.5 mM Tris-HCl pH 6.8, 0.5 M β-mercaptoethanol, 2% SDS, 10% glycerol, 0.01% bromophenol blue). Quantification of RIPA extracts was performed using the Thermo Fisher Scientific Pierce BCA Protein Assay kit. Protein transfer was performed by wet transfer using 1× Towbin buffer (25 mM Tris, 192 mM glycine, 0.01% SDS, 20% methanol) and nitrocellulose membrane. Blocking was performed in 5% milk in 1× TBST (19 mM Tris, 137 mM NaCl, 2.7 mM KCl and 0.1% Tween-20) for 1 h at room temperature. The following antibodies were diluted in 1% milk in 1× TBST: anti-APOBEC3A/B/G (04A04) and anti-APOBEC3A (01D05) (see below; western blot, 1:1,000), anti-APOBEC3B (Abcam; ab184990; western blot, 1:500), anti-REV1 (Santa Cruz; sc-393022, western blot, 1:1000), anti-SMUG1 (Abcam; ab192240; western blot, 1:1,000 and Santa Cruz; sc-514343; western blot, 1:1,000), anti-UNG (abcam; ab109214; western blot, 1:1,000), anti-GFP (Santa Cruz; sc-9996; western blot, 1:1,000), anti-β-actin (Abcam; ab8224; western blot, 1:3,000), anti-β-actin (Abcam, ab8227; western blot, 1:3,000); anti-mouse IgG HRP (Thermo Fisher Scientific; 31432; 1:10,000), anti-rabbit IgG HRP (SouthernBiotech; 6441-05; 1:10,000).
APOBEC3 monoclonal antibody generation
Residues 1–29 (N1-term) or 13–43 (N2-term) from APOBEC3A and residues 354–382 (C-term) from APOBEC3B and were used to create three peptide immunogens (EZBiolab). Five mice were given three injections using keyhole limpet haemocyanin (KLH)-conjugated peptides over the course of 12 weeks (MSKCC Antibody and Bioresource Core). Test bleeds from the mice were screened for anti-APOBEC3A titres by enzyme-linked immunosorbent assay (ELISA) against APOBEC3A peptides conjugated to BSA. Mice showing positive anti-APOBEC3A immune responses were selected for a final immunization boost before their spleens were collected for B cell isolation and hybridoma production. Hybridoma fusions of myeloma (SP2/IL6) cells and viable splenocytes from the selected mice were performed by the MSKCC Antibody and Bioresource Core. Cell supernatants were screened by APOBEC3A ELISA. The strongest positive hybridoma pools were subcloned by limiting dilution to generate monoclonal hybridoma cell lines. The hybridomas 04A04 (anti-APOBEC3A/B/G) and 01D05 (anti-APOBEC3A) were expanded then grown in 1% FBS medium. This medium was clarified by centrifugation and then passed over a protein G column (04A04) or protein A column (01D05) to bind to monoclonal antibodies. The resulting monoclonal antibodies were eluted in PBS (04A04) or in 100 mM sodium citrate pH 6.0, 150 mM NaCl buffer and subsequently dialysed into PBS (01D05).
Cell cycle and apoptosis assays
Annexin V staining was performed using the annexin V Apoptosis detection kit (BD Biosciences) according to the manufacturer’s instructions. For propidium iodide plus BrdU double staining, BrdU was added to the culture medium to a final concentration of 10 μM for 1 h. Cells were fixed with 70% ethanol and treated with 2 M hydrochloric acid for 20 min. BrdU staining was performed with 20 μl of anti-BrdU antibodies (25 μg ml−1, B44, Becton Dickinson) for 15 min at room temperature followed by a 15 min incubation with 50 μl Alexa Fluor 488 goat anti mouse at 40 μg ml−1 (Invitrogen). After a final wash, cells were taken up in 100 μg ml−1 PI with 20 μg ml−1 RNase A. Flow data were collected on the Fortesa or LSR-II analyzer and analysed using FlowJo v.10.
Automatic counting of γH2AX foci
EdU staining was performed by using Click-iT EdU Alexa Fluor 488 Imaging Kits (Invitrogen, C10337) according to the manufacturer’s instructions. For EdU incubation, EdU was added to the culture medium to a final concentration of 10 μM for 2 h. Cells were fixed with 2% paraformaldehyde for 15 min at room temperature followed by 0.5% Triton X-100 permeabilization for 5 min. Click-iT reaction was performed according to the manufacturer’s instructions. γH2AX was stained with anti-γH2AX antibodies (EMD Millipore, 05-636-1, 1:1,000) for 2 h at room temperature followed by anti-mouse secondary antibody Alexa Fluor 647 (Invitrogen, A21235). Cells were stained with Hoechst (1 μg μl−1) and mounted with Prolong Gold Antifade Reagent (Invitrogen, P36934). Images were acquired on the DeltaVision Elite system equipped with a DV Elite CMOS camera, microtitre stage, and ultimate focus module (z stack through the cells at 0.2 mm increments). All of the images were processed by maximal projection of the z stack image…
