Cell culture
Table of Contents
Culturing of cell lines
MDA-MB-231 (ATCC, CRM-HTB-26), HEK 293T/17 (ATCC, CRL-11268) and BJ-5ta (ATCC, CRL-4001) cells were obtained from the American Type Culture Collection (ATCC) and were cultured according to the manufacturer’s specifications HEK 293T/17 cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Corning, 10-013-CV) supplemented with 10% FBS (VWR, 76294-120). Triple-negative MDA-MB-231 cells were cultured in minimum essential medium (MEM) (Gibco, 11095-080) supplemented with 1% sodium pyruvate (VWR, 97061-448) and 10% FBS. The hTERT-immortalized human dermal fibroblast BJ-5ta cells were cultured in DMEM supplemented with 10% FBS and 0.01 mg ml–1 hygromycin B (Sigma-Aldrich, H7772). RAW-Lucia ISG cells (Invivogen, rawl-isg) were grown in DMEM with 10% FBS and 100 µg ml–1 Normocin (Invivogen, ant-nr-05). At every other passage, 200 µg ml–1 of Zeocin (VWR, AAJ67140-XF) was added for selection. All cells were cultured at 37 °C in a humidified incubator with 5% CO2 in the air and were tested monthly for mycoplasma using a PlasmoTest kit (Invivogen, rep-pt1).
Cell line identification
In accordance with the guidelines set by the International Cell Line Authentication Committee, we verified the identity of all cell lines used in this study. The cell lines were tested by Genetica DNA Laboratories. For human cell lines, the PowerPlex16HS method was used, which includes a mouse marker for the detection of mouse DNA. For mouse cell lines, the mouse STR method was adopted, incorporating two human markers for detecting human DNA. The results from these tests confirmed the identity of each cell line, ensuring their authenticity. The last authentication test was conducted on 10 August 2023.
Collection of pMMECs
pMMECs were derived by collecting the fourth and fifth mammary glands from 8–12-week-old virgin female transgenic mice with the desired genotype. pMMECs were isolated without performing randomization. Glands were incubated in Liberase digestion medium ((EpiCult-B Mouse Medium kit, StemCell Technologies, 05610), 150 U ml–1 collagenase type 3 (Worthington, LS004182), 20 mM HEPES (Thermo Fisher Scientific, 15630106) and 20 µg ml–1 Liberase Blendzyme 2 (Roche, 11988425001)) and shaken vertically at 37 °C overnight. The resulting digestion was spun down and resuspended in trypsin (Gibco, 25200056) with DNase I (Worthington, LS002060) and incubated at 37 °C for 5 min. DMEM with 5% FBS was added to neutralize the trypsin. Cells were spun down and resuspended in dispase (Stem Cell Technologies, 07913) and DNase I and incubated at 37 °C for 5 min. Cells were washed twice with DMEM, and the resulting cells were resuspended in Epi-Cult medium and seeded onto plates coated with Cultrex 3D culture matrix rat collagen I (Fisher Scientific, 3447-020-01).
Collecting tumour-derived pMMECs
For mammary tumour induction, 6–12-week-old virgin female R26cas9/cas9, MycOE and Trp53fl/fl mice received bilateral intraductal injections into the fourth mammary gland containing 5 × 105 TU of the indicated LentiCRISPR-Cre-V2-sgRNA lentivirus. Mouse cohorts were palpated for the development of mammary tumours. The mice were regularly examined twice a week for the development of mammary tumours. After the tumours had formed, they were examined three times a week. The mice were euthanized in a humane manner in accordance with the guidelines set by the UNC Institutional Animal Care and Use Committee (IACUC) when they reached a predetermined experimental end point. The mammary tumours were then collected during necropsy and divided into four pieces for further analysis. Tumour pMMECs were isolated without performing randomization. One of the tumour samples was incubated in digestion medium (DMEM with 10% FBS, 1 mg ml–1 collagenase type 3 and 1 mg ml–1 hyaluronidase (Worthington, LS005477)) and shaken horizontally at 37 °C for 4 h. The digestion mix was spun down and resuspended with trypsin containing DNase I at 37 °C for 5 min. DMEM was added to neutralize the trypsin. Cells were spun down and incubated in dispase and DNase I at 37 °C for 5 min. Cells were washed with DMEM and passed through a 70 μm filter. Tumour cells were resuspended in HuMEC Ready Medium (1X) (Thermo Fisher Scientific, 12752010) medium.
MRE11 nuclease inhibition assay
The MRE11 inhibitors mirin (Sigma-Aldrich, M9948), PFM39 (Sigma-Aldrich, SML1839) and PFM01 (Sigma-Aldrich, SML1735) were administered at a concentration of 100 µM unless otherwise specified. This was followed by the transfection of biotin-conjugated 90-mer dsDNA (biotin-ISD90) at a concentration of 200 pmol, 30 min after application of the MRE11 inhibitor. Cells were then collected for western blotting 3 h after dsDNA transfection. The localization of cGAS and biotin-ISD90 was detected using immunocytochemistry (ICC) 2 h after dsDNA transfection, with biotin-ISD90 detected using streptavidin–Alexa 488 (Thermo Fisher Scientific, S32354). Last, for quantitative PCR (qPCR) analysis, cells were collected 6 h after dsDNA transfection.
Transgenic mouse models
All animal experimentation was conducted with approval by the UNC IACUC. R26LSL-cas9 (JAX, 024857) and R26LSL–MycOE (JAX, 020458) transgenic mouse strains were obtained from the Jackson Laboratory. Trp53fl/fl mouse strains were provided by the Perou Laboratory and originally obtained from the Frederick National Laboratory for Cancer Research (strain 01XC2). R26cas9/cas9, MycOE and Trp53fl/fl lines were used in the in vivo DDR-CRISPR screen. A subset of interbred mouse strains used in this study were analysed using a Mouse Universal Genotyping Array from Neogen Genomics and determined to be >90% FVB with a minor contribution from C57BL/6J. Blinding of animal experiments was not performed.
Housing conditions for mice
At the Division of Comparative Medicine, the standard light cycle for mice is set from 07:00 to 19:00. The ambient temperature for rodents is maintained between 20 and 23 °C, with a humidity level ranging from 30% to 70%. When setting up breeder cages, nesting material was added, and the types of nesting materials that can be used are outlined in the IACUC’s Environmental Enrichment Program for Animals. We documented the date of birth of the mice and recorded it on the Division of Comparative Medicine’s Weaning Notice within 3 days of their birth. For non-breeding cages, a maximum of five adult mice of the same sex were housed per cage. Pups are typically weaned by the time they are 23 days old, especially when their weight reaches around 10 g. However, if a pup weighs less than 10 g by the 23rd day, exceptions to this guideline are considered.
In vivo DDR-CRISPR screen
A total of 40 virgin female transgenic mice of R26cas9/cas9, MycOE and Trp53fl/fl lines aged 6–10 weeks were intraductally injected with 10 μl lentivirus generated from the Lenti-CRISPR-Cre-V2-sgRNA DDR Library plasmid at 5 × 105 TU into the fourth mammary glands bilaterally. Mice were selected for the experiment without randomization. Mice were palpated twice a week for tumour detection. Two mice each were collected at 1, 3, 6, 9 and 12 weeks to assess for mammary hyperplasia and to stain for GFP to assess viral infectivity. Mice with a single tumour were euthanized for collection when the tumour reached a maximum diameter of 2.0 cm. If bilateral tumours were present, the mouse was euthanized when the largest tumour reached a diameter of 1.5 cm. Mice were euthanized in compliance with IACUC protocols. At the time of necropsy, abdominal exploration was performed for gross liver metastases and thoracotomy was performed for gross lung metastases. Splenic tissue was collected and banked for gDNA collection. Each mammary tumour was sectioned into four pieces, with two pieces flash-frozen for RNA and DNA extraction. One piece was fixed in 4% paraformaldehyde (PFA) for tissue processing and staining. The remaining piece was taken for the creation of the mammary tumour cell line. sgRNA abundance was determined from flash-frozen samples to eliminate effects of tissue culture variances. Sample randomization was not performed.
Establishment of stable cell lines and viral production and infection
Generation of cell lines expressing a hypomorphic Mre11 mutation
crRNA was designed using MIT CRISPR (crispr.mit.edu) to target codon 633 of Mre11, which is the site of a truncating mutation that gives rise to the radiosensitive ataxia-telangiectasia-like disorder. We performed Neon transfection (Invitrogen, MPK1025) with recombinant Cas9 purified by the University of North Carolina Center for Structural Biology Protein Expression and Purification Core), crRNA (Integrated DNA Technologies (IDT), customized) and tracrRNA (IDT, 1072532) using the manufacturer’s protocol and electroporation settings. Forty-eight hours after transfection, cells were seeded for single-clone selection. Restriction enzyme screening, western blots, PCR screening and Sanger sequencing confirmed gene targeting and functional assays.
Halo-conjugated mutant cell lines
Retroviral pBABE-puromycin-Halo vectors (gift from E. Rothenberg, NYU) encoding the desired construct (15 µg) were co-transfected with pUMVC (10 µg) (Addgene, 8449) and pCMV-VSV-G (2 µg) (Addgene, 8454) into a 10 cm diameter dish of 70% confluent HEK 293T/17 cells to generate retrovirus. In brief, plasmids were mixed with 30 µl Lipofectamine 3000 (Thermo Fisher Scientific, L3000001) in 1.0 ml Opti MEM reduced serum medium (Thermo Fisher Scientific, 31985062) and incubated at room temperature for 20 min. The transfection mix was then added dropwise to a 10 cm dish. After 16 h of transfection, the transfection mix was removed, and 15 ml of fresh medium was added to the cells. Twenty-four hours later, the cell culture medium was collected, centrifuged for 5 min at 1,000g and the viral supernatant filtered using 0.45 µm sterile syringe filters. Next, 60% confluent target cells were seeded in a 6-well plate and were infected with 2 ml of the retroviral medium containing 8 µg ml–1 hexadimethrine bromide–polybrene (Sigma-Aldrich, 107689) for 24 h. Medium containing 2 µg ml–1 puromycin (Thermo Fisher Scientific, A1113802) was used for selection of cells that had integrated the constructs. A pool of transduced cells was utilized for subsequent experiments following complete death of non-transduced cells placed under selection in parallel.
Cells lines for live-cell imaging
The lentiviral vector pTRIP-CMV-tagRFP-Flag-cGAS (Addgene, 86676) or pTRIP-CMV-GFP-Flag-cGAS (Addgene, 86675) (12 µg) was co-transfected with 6 μg psPAX2 (Addgene, 12260) and 3 μg pMD2.G (Addgene, 12259) to 80% confluent HEK 293T/17 cells in a 10 cm culture dish. In brief, plasmids were mixed with 42 µl polyethylenimine (PEI; 1 mg ml–1 linear PEI, Sigma Aldrich, 765090) and incubated at room temperature for 20 min. The transfection mix was then added dropwise to a 10 cm dish. After 16 h of transfection, the medium was removed and 15 ml of fresh medium was added to the cells. Twenty-four hours later, the cell culture medium was collected for 3 days, centrifuged for 5 min at 1,000g and filtered using 0.45 µm sterile syringe filters. Next, 60% confluent target cells were seeded in a 6-well plate and were infected with titred lentiviral medium containing 8 µg ml–1 polybrene for 24 h. Medium was replaced every 24 h for 2 days. Cells were seeded for single-clone selection in a 96 well-plate. Single clones were selected for Sanger sequencing and functional assays for relevant genes.
To generate knockouts of indicated genes in pMMECs, HEK 293T/17 cells were transfected using PEI with the viral packaging plasmids psPax2 and pMD2.G and with LentiCRISPR-Cre-V2-sgRNA-Lumifluor harbouring sgControl, sgMRE11, sgcGAS, sgSTING, sgZBP1 (1, 2 and 3), sgMLKL, sgRIPK3 or sgATM plasmids. After 16 h of transfection, medium was removed, and 15 ml of fresh medium was added to the cells. Twenty-four hours after transfection, the cell culture medium was collected for 3 days, centrifuged for 5 min at 1,000g and filtered using 0.45 µm sterile syringe filters. Medium containing virus was spun down for 2 h at 16 °C at 21,000 r.p.m. The virus-containing pellet was resuspended in 1:100 the initial volume of PBS and incubated at 4 °C for 24 h then aliquoted and stored at −80 °C until use. Virus aliquots were thawed immediately before use to avoid loss of viral transduction efficiency with repeated freeze–thaw cycles. For lentiviral infections, cells were transduced with the appropriate virus combined with 8 µg ml–1 polybrene overnight. Cells were refed with virus-containing medium and incubated for 2 days. Following the last infection, cells were washed three times with PBS and cultured with HuMec medium. For testing viral efficacy, a small sample of cells were fixed with 3% PFA and were assessed by flow cytometry (Attune NxT, UNC Flow Cytometry Core) for the presence of GFP (indicating Cas9 expression), mCherry (indicating PCNA expression) or were stained with anti-CD2-PE (indicating MYC expression).
To generate cGAS knockout MDA-MB-231 cell lines, synthetic gRNAs targeting human cGAS were purchased from Synthego (CRISPR gene knockout kit V2). gRNAs were diluted to 30 µM (pmol µl–1) in 1× TE buffer (Synthego). Cas9 (Synthego) was diluted to 20 µM (pmol µl–1) in 1× TE buffer. These were stored at −80 °C until use. A sgRNA to Cas9 ratio of 3:1 was used for RNP formation. Next, 3 µl (90 pmol) of sgRNA, 0.5 µl of 10 pmol Cas9 and 3.5 µl resuspension buffer were added to a 1.5 ml tube per sample. The RNP complexes were allowed to incubate for at least 10 min at room temperature. Cells were subcultured 2 days before electroporation and seed cells were cultured in an appropriately sized vessel so that they are 70–80% confluent on the day of transfection. Each electroporation reaction used approximately 1 × 105 cells. Next, 1 ml of pre-warmed MEM medium was added into each well of a 12-well plate per reaction. RNP complex (7 µl) was added to 5 µl of MDA-MB-231 cell suspension to make 12 µl of cell–RNP solution per reaction and electroporated using a Neon Transfection system (Thermo-Fisher, MPK5000) at a pulse code (20 ms × 1 pulse) using 10 µl Neon tips (Thermo-Fisher, MPK1025) at various voltages (1,800–2,200 V). In knockout experiments, cell–RNP solutions were electroporated at 20 ms × 4 pulse and 1,400 V. Immediately following electroporation, cells were slowly pipetted into pre-warmed culture medium (MEM supplemented with 1% sodium pyruvate and 10% FBS) in a 12-well plate. Cells were then incubated at 37 °C. Cells were cultured for 3–6 days following electroporation before reading out gene-editing efficiency by flow cytometry or Sanger sequencing.
Generation of stable doxycycline-inducible human cGAS MDA-MB-231 cell lines
We used a piggyback system to generate two stable doxycycline-inducible human cGAS MDA-MB-231 cell lines. First, we obtained cGAS WT conjugated with the attB sequences by PCR using custom-designed primers (IDT, forward: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGCAGCCTTGGCACGGAAAG-3′; reverse: 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAAAATTCATCAAAAACTGG-3′) from a pTRIP-CMV-tagRFP-Flag-cGAS vector. Then, we subcloned it into pDONR 221 (Thermo Fisher Scientific, 12536017) using Gateway BP Clonase II enzyme mix (Thermo Fisher Scientific, 11789100), followed by cloning into the PB-TA-ERP2 (Addgene, 80477) destination vector the LR Clonase II enzyme mix (Thermo Fisher Scientific, 11791100) reaction according to the manufacturer’s protocol, resulting in the generation of PB-TA-ERP2-hcGAS-WT, and transformed into competent cells. We then performed site-directed mutagenesis of cGAS residues that engage the nucleosomal AP, in which R255 was replaced by alanine (R255A). Primers were designed and ordered (IDT, forward: 5′-ATTTAAAGCAAATCCGAAAGAA-AATCCTCTGA-3′; reverse: 5′-GGATTTGCTTTAAATTTCACAAAGTAATATGCA-3′) and PCRs performed. PCR products were confirmed by gel electrophoresis, and purified PCR products were incubated with 20 U of Dpn1 (NEB, R0176S) to digest the template DNA. The amplified plasmid was used for transforming competent cells (Lucigen, 60241-2). cGAS genes on amplified plasmids (PB-TA-ERP2-hcGAS-R255A) were sequenced (Plasmidsaurus) to confirm that the mutation had been introduced at the correct place and that no additional mutation occurred during the PCR process. Cells were seeded in a 6-well plate at a density of 100,000 cells per well. piggyBac vectors (PB-TA-ERP2-hcGAS-WT or PB-TA-ERP2-hcGAS-R255A) were co-transfected with a piggyBac transposase vector (System Biosciences, PB210PA-1). DNA was transfected at a transposon to transposase ratio of 1 µg:200 ng cGAS knockout MDA-MB-231 cell lines using Lipofectamine 3000 following the manufacture’s protocol. Three days after transfection, cells were selected with 2 µg ml–1 puromycin. For expression assays, cells were induced with doxycycline hyclate (Sigma-Aldrich, D9891) at a final concentration of 1 µg ml–1 for 24 h.
Cloning
LentiCRISPR-Cre-V2-sgRNA LumiFluor plasmids
This plasmid was created using restriction enzymes (XbaI and BglII) to cut the Cre sequence from the pLV-Cre_LKO1 plasmid (Addgene, 25997) and swapping it for the Cas9 sequence in lentiCRISPR V2 (Addgene, 52961) through restriction digest and T4 ligation. To remove the BsmbI site within Cre, Gibson cloning was used (HiFi DNA Assembly master mix; NEB, E2611S) to change the sequence of a valine residue from GTC to GTA, thus removing the site while preserving the protein sequence. The Lumifluor construct was cloned using a Lentiviral_pRRL-EF1a-GpNLuc plasmid (gift from A. Amelio) as previously described39. Using the remaining BsmB1 sites, our custom DDR-CRISPR library sequences (containing 3,908 sgRNAs targeting 309 mouse DDR genes with an average of 10 sgRNAs per gene, as well as 834 non-targeting sgRNA controls) was inserted into the sgRNA scaffolding region. The same process was used to generate LentiCRISPR-Cre-V2-sgControl Lumifluor plasmid (Trp53bp1 intronic sequence on chromosome 2), LentiCRISPR-Cre-V2-sgMRE11 Lumifluor plasmid (targeting codon 633 of MRE11) and additional plasmids targeting cGAS, STING, ZBP1, RIPK3 and MLKL. All plasmids created were confirmed by Sanger sequencing (Eton Bioscience).
pBABE-Puro-Halo-MRE11 mutant plasmids
dsDNA molecules (gBlocks Gene Fragments) were ordered from IDT to design DNA constructs with the BstBI region added to the human MRE11(H129N) or DB1 and DB2 deletion fragment. The gBlocks and pBABE-Puro-Halo vector were then cut using the BstBI restriction enzyme (NEB, R0519S). Samples were resuspended in nuclease-free distilled water (IDT, 11-05-01-04) to a concentration of 10 ng µl–1. Samples were heated at 50 °C for 20 min per the manufacturer’s instructions and stored at −20 °C. Finally, the MRE11(H129N) and MRE11(DB1Δ/DB2Δ) sequences were inserted into the pBABE-Puro-Halo vector using Quick DNA ligase (NEB, M2200S).
pBig1a-hRAD50-hMRE11N2×STR plasmids
pFastBac1 plasmids containing genes for recombinant human MRE11–Flag (pTP813, Addgene, 113308) and RAD50–6×HIS (pTP2620, Addgene, 113311)40,41 were provided as gifts from T. Paull. The pBig1a-hRAD50-hMRE11N2×STR plasmid (human RAD50 untagged, human MRE11 with a C-terminal TEV protease cleavage site (N) followed by a twin-strep tag (2×STR)) was cloned by Gibson assembly as previously described42. In brief, hMRE11N2×STR was cloned into the pLIB vector by Gibson assembly using pTP813 as a template. pLIB-hMRE11N2×STR and pTP2620 were then used as templates to prepare pBig1a-hRAD50-hMRE11N2×STR by Gibson assembly, which was confirmed by Sanger sequencing (Azenta Life Sciences). The nuclease-dead MRE11(H129N) mutation was incorporated by site-directed mutagenesis (Q5 Hot Start High-Fidelity 2× master mix, NEB), creating pBig1a-hRAD50-hMRE11(H129N)N2×STR, which was confirmed by whole plasmid sequencing (Plasmidsaurus).
Transfections
siRNA transfection
Pre-designed siRNAs were purchased from Dharmacon: negative Control scramble siRNA (4390847); and siRNA directed against human targets MRE11 (M-009271-01), RAD50 (L-005232-00) and NBN1 (L-009641-00). Cells were transfected with siRNAs using Lipofectamine RNAimax reagent (Thermo Fisher Scientific, 13778075) according to the manufacturer’s instructions. In brief, MDA-MB-231 cell lines plated on 6-well plates at a density of 200,000 cells per well for siRNA treatment. Twenty-four hours after plating, cells were exposed to 30 nM per well siRNAs in Opti-MEM with Lipofectamine RNAimax reagent. Forty-eight hours after transfection, cells were transfected with 200 pmol of 90-mer dsDNA (ISD90), Oregon-green hNCP (OG-hNCP)-v139 or hNCP-v52 using Lipofectamine 2000 reagent (Thermo Fisher Scientific, 11668019) according to the manufacturer’s instructions.
DNA or nucleosome transfection
A total of 500,000 cells were seeded on an 18 mm square cover glass in a 6-well plate. Twenty-four hours after plating, Lipofectamine 2000 reagent (10 µl) in 250 µl Opti-MEM per transfection were incubated with indicated concentrations of ISD90 or nucleosomes per reaction in 250 µl Opti-MEM for 15 min at room temperature before the addition of mix (500 µl) to the cells. Cells were then fixed or collected for imaging or western blotting at each time point.
Quiescent assay (EdU incorporation)
pMMECs were infected with a lentivirus containing sgRNA and Cre. Between 5 and 9 days after viral infection, cells were reseeded on coverslips in 6-well plates. These cells were then incubated with EdU for 24 h, fixed in 4% formaldehyde for 20 min and permeabilized in 0.5% (v/v) Triton X-100 for another 20 min. EdU detection was performed using an EdU detection kit (Sigma-Aldrich, BCK-EDU594) according to the kit’s protocol.
The cells were then blocked in a solution of 3% BSA in PBS for 30 min and subsequently incubated in DAPI for 10 min. The coverslips were mounted onto slides using a mounting solution (Thermo Fisher Scientific, P36934). EdU-negative cells were examined using an EVOS M7000 fluorescence microscope.
For the quiescence assay involving drug or chemical treatment, cells were incubated with or without 25 µl 2′3′-cGAMP (Invivogen, tlrl-nacga23-1), 0.5 µM C-176 (Selleck Chemicals, S6575), 1 µM NSA (Selleck Chemicals, S8251) or 10 µM HS-1371 (Selleck Chemicals, S8775) for 6 h before the EdU treatment.
Determining the frequency of micronuclei
After fixation and staining with DAPI, the percentage of cells with micronuclei was determined using either a fluorescence microscope (Olympus BX61 or EVOS M7000) or confocal microscopes (LSM710) under blinded conditions. Micronuclei were defined as discrete DNA aggregates that were separate from the primary nucleus and were only identified in cells in which the interphase primary nuclear morphology was normal. Cells exhibiting an apoptotic appearance were excluded from this analysis.
Immunofluorescence
ICC analysis
Cells were fixed in ice-cold methanol for 10 min at −20 °C. Fixed cells were pre-incubated in blocking solution (3% BSA in PBS) followed by incubation with primary antibodies at 4 °C overnight. After incubation with primary antibodies, cells were washed three times with shaking in PBS and probed with fluorescein-conjugated (Cy3, Cy5, Alexa 488, Alexa 549 and Alexa 674) anti-mouse or anti-rabbit secondary antibodies. After washing 3× with PBS, DAPI was used for DNA counterstaining followed by mounting on slides. To determine the localization of cGAS in the cytoplasm or nucleus, cells were fixed in 3.8% formaldehyde for 15 min at room temperature. The cells were then permeabilized in 0.2% Triton X-100 for 10 min at room temperature. Fluorescence images were taken using an EVOS M7000 or Olympus BX61 or a Zeiss LSM710 spectral confocal laser scanning microscope at the UNC Microscopy Service Laboratory. Image analysis was performed using ImageJ.
The following primary antibodies were used for immunofluorescence: anti-cGAS mouse-specific (Cell Signaling, 31659; 1:500); anti-cGAS (Cell Signaling, 15102; 1:500); anti-α-tubulin (Santa Cruz Biotechnology, sc-5286; 1:500); and anti-MLKL phosphor S345 (Abcam, ab196436; 1:500). The following secondary antibodies were used (at 1:500 dilution): anti-rabbit-Alexa 488 (Thermo Fisher Scientific, A11034); anti-rabbit-Alexa 594 (Thermo Fisher Scientific, A11037); anti-rabbit-Alexa 633 (Thermo Fisher Scientific, A21072); anti-mouse-Cy3 (Jackson ImmunoResearch, 715-165-151); anti-rabbit Cy5 (Jackson ImmunoResearch, 111-175-144); and streptavidin–Alexa 488 (Thermo Fisher Scientific, S32354).
All subsequent analyses and processing of images were performed using ZEN 2011 microscope software (Zeiss) or ImageJ software.
cGAS/pH2A.X staining for tumour immunofluorescence
Sequential dual immunofluorescence was performed on paraffin-embedded tissues that were sectioned at 5 µm. The immunofluorescence assays were performed using a Bond automated slide staining system (Leica Microsystems) with a Bond Research Detection System kit (Leica, DS9455). cGAS (31659S) and phospho-histone H2A.X (9718s) were both purchased from Cell Signaling Technology. Slides were deparaffinized in Bond dewax solution (Leica, AR9222), hydrated in Bond wash solution (Leica, AR9590) and sequentially stained for cGAS and then pH2A.X. Specifically, antigen retrieval for cGAS was performed for 20 min at 100 °C in Bond-epitope retrieval solution 2 pH 9.0 (Leica, AR9640). After pretreatment, slides were incubated for 1 h with cGAS antibody (1:4,000) followed by Novolink Polymer (Leica, RE7161) then TSA Cy5 (Akoya Biosciences, FP1117). Afterwards, a second round of antigen retrieval was performed for 20 min at 100 °C in Bond-epitope retrieval solution 1 pH 6.0 (Leica, AR9961). Slides were then incubated with the phospho-histone H2A.X antibody (1:3,000, 2 h) then Novolink Polymer and detected using TSA Cy3 (Akoya Biosciences, FP1046). Nuclei were stained using Hoechst 33258 (Invitrogen, H3569). The stained slides were mounted using ProLong Gold antifade reagent (Life Technologies, P36930). Positive and negative controls (no primary antibody) were included in this run. Single-stain controls were done for multiplex immunofluorescence stains for which one primary antibody was omitted to ensure that cross-reactivity between the antibodies did not occur. Slides were then digitalized using Aperio ScanScope FL (Aperio Technologies). The digital images were captured in each channel using a ×20 objective (0.468 µm per pixel resolution) using line-scan camera technology43. The adjacent 1 mm stripes captured across the entire slide were aligned into a contiguous digital image by an image composer. Slides were scanned on a Versa slide scanner (Leica Biosystems) using a ×40 objective (0.16276 µm per pixel resolution; MPP), with an image bit depth of 8 bits per channel. Images were analysed using ImageScope software and ImageJ.
phospho-histone H3 staining for tumour immunofluorescence
Chromogenic immunohistochemistry (IHC) was performed on paraffin-embedded tissues that were sectioned at 5 µm. IHC was carried out using a Bond III Autostainer system (Leica Microsystems). Slides were dewaxed in Bond dewax solution and hydrated in Bond wash solution. Heat-induced antigen retrieval was performed for 20 min at 100 °C in Bond-epitope retrieval solution 1 pH 6.0. After pretreatment, slides were incubated with phospho-histone H3 (Sigma-Aldrich, 06-570) at 1:1,000 for 1 h followed by Novolink Polymer (Leica, RE7260-K). Antibody detection with 3,3-diaminobenzidine (DAB) was performed using a Bond Intense R detection system (Leica, DS9263). Stained slides were dehydrated and coverslipped with Cytoseal 60 (Thermo Fisher Scientific, 8310-4). A positive control and a negative control (no primary antibody) were included for this run. IHC-stained slides were digitally imaged using an Aperio ScanScope AT2 (Leica Biosystems) with a ×20 objective.
Live-cell imaging
Time-lapse analysis of pMMECs
For cell cycle time-lapse studies, pMMECs were collected from R26cas9/cas9, MycOE, and Trp53fl/fl virgin female mice aged 8–12 weeks and plated on collagen-coated 6-well plates at a minimum density of 50,000 cells per well using the pMMEC collection protocol. pMMECs were isolated without performing randomization. At 4 days after collection, pMMECs were transduced with 1 μl of each lentivirus per well (PCNA–mCherry and Cre-sgControl-Lumifluor or PCNA–mCherry and Cre-sgMRE11-Lumifluor) at 1–5 × 105 TU using 4 µg ml–1 polybrene daily for 2 days23. On day 7, cells were washed using EpiCult basal medium before being transferred to 12-well glass bottom plates (Cellvis, P12-1.5H-N) coated with Cell Tak (Corning, 354240), and LA7s (ATCC, CRL-2283) were lethally irradiated at 70 Gy ionizing radiation using a Bio-Rad Source RS2000 irradiator (250,000 cells per well to create a confluent feeder monolayer). pMMECs were seeded at a density of 1 × 105 per well in LA7 medium (DMEM with reduced sodium bicarbonate (1.5–2.0 g l–1), 4.5 g l–1 glucose (Thermo Fisher Scientific, 11965084), 0.005 mg ml–1 insulin (VWR, 11061-68-0), 20 mM HEPES and 5% FBS) for the co-culture. On day 10, LA7 medium was replaced with imaging-optimized LA7 medium (made with Phenol Red-free DMEM:F12 medium, Gibco 21041-025). Images of cells were captured every 20 min for 72 h in the mCherry and GFP fluorescence channels at a constant temperature of 37 °C and atmosphere (5% CO2). Fluorescence images were obtained using a Nikon Ti Eclipse inverted microscope with a ×40 objective and a Nikon Perfect Focus system to maintain acquisition focus. Nikon’s NIS Elements AR software was utilized for image acquisition. Image analysis was performed using ImageJ. Cells positive for both GFP (indicating Cas9 activation) and mCherry were analysed in the imaging dataset.
Imaging of stable cell lines
cGAS recruitment over time after transfection with OG-hNCP was analysed in MDA-MB-231 WT and MRE11-deficient stable cell lines expressing RFP–cGAS. Cells were plated into 12-well glass-bottom plates (Cellvis, P12-1.5H-N) supplemented with 10% FBS and sodium pyruvate. Twenty-four hours after plating, cells were transfected with OG-hNCP, then images were captured. Microscopy images were obtained using a Nikon Ti2 widefield microscope equipped with a Plan Apo ×20 (0.75 NA) air objective. Throughout the imaging experiments, cells were maintained at 37 °C and 5% CO2 using an Okolab H301-PI-736-160×110 live cell imaging chamber. A total of two fields of view per condition were imaged every 14.5 s. All images were obtained using a Lumencor SPECTRA Light Engine for illumination, a motorized emission filter turret and a Hamamatsu Orca-Flash4.0 camera. During each round of image acquisition, four successive images (Brightfield, Halo-JF646-MRE11, RFP–cGAS and OG-hNCP) were obtained. Nikon’s NIS Elements AR software was utilized for image acquisition. Image analysis was performed using ImageJ.
FRAP assays
For FRAP assays, MDA-MB-231 cells expressing GFP–cGAS were grown in a 4-well glass-based dish (Thermo Scientific, 155382PK). Cells were transfected with siControl or siMRE11 for 60 h, then FRAP assays were performed on a LSM710 confocal microscope. All experiments were carried out at 37 °C, and imaging was performed with a ×63 oil-objective lens using the bleaching mode of the Zeiss software. For each experiment, five pre-bleach images were taken, and a single ROI spot was bleached with the 405 nm line at 20% transmission. To gain the full stack of focal planes during recoveries, the pinhole of 488 nm was adjusted to 384 nm. Then, images were collected over a period of 60 min every 1 min. Image analysis was performed using ZEN 2011 (Zeiss) and ImageJ software. Fluorescence images were taken using a Zeiss LSM710 spectral confocal laser scanning microscope at the UNC Microscopy Service Laboratory. Image analysis was performed using ImageJ.
Growth assays
pMMECs were infected with sgControl, sgMre11, sgcGas or sgSting Cre-lenti-viruses. Five days after infection, the cells were seeded into 12-well plates at a density of 3 × 104 cells per well. Duplicate samples were then collected every 2–3 days for a period of 15 days. The total number of cells per well was counted, and the cells were fixed in 3% PFA. Following fixation, the cells were subjected to flow cytometry analysis using an Attune NxT flow cytometer to detect the presence of GFP.
cGAMP ELISA
For 2′3′-cGAMP quantification, cells were collected by trypsinization using 0.05% trypsin EDTA (Gibco, 25300054) for 5 min. Cell pellets were then lysed in RIPA lysis buffer containing 50 mM Tris, 150 mM NaCl, 1% (w/v) sodium deoxycholate, 0.03% (v/v) SDS, 0.005% (v/v) Triton X-100, 5 mM EDTA, 2 mM sodium orthovanadate and complete protease inhibitor cocktail (Roche). The pellet from 1 well of a 6-well plate was resuspended in 130 µl of RIPA lysis buffer and incubated for 30 min on ice. The lysed cells were then centrifuged for 10 min at 18,200g and 4 °C. The supernatant (100 µl) was subsequently used for the cGAMP ELISA assay (Cayman, 501700) as per the manufacturer’s instructions. Protein concentration in the supernatant was measured using a Qubit 3.0 fluorometer system, which was then used to normalize the 2′3′-cGAMP levels.
Immunoblotting
Cell pellets were lysed in 2× Laemmli sample buffer (Bio-Rad, 161-0737) containing 4% β-mercaptoethanol. The samples were then heated to 95 °C for 5 min, and protein concentrations were determined using a Qubit 3.0 fluorometer (Thermo Fisher Scientific, Q33216) according to the manufacturer’s instructions. Approximately 10–30 µg of total protein was separated on SDS–PAGE gels (4–20% (Mini-PROTEAN TGX, Bio-Rad), 10% or 15% (v/v)). The gels were then transferred onto PVDF membranes (Bio-Rad, 1704272) using a Trans-Blot Turbo transfer system (Bio-Rad, 1704270).
The membranes were briefly washed in Tris-buffered saline-Tween 20 (TBST) (50 mM Tris-Cl, pH 7.5, 150 mM NaCl and 0.1% (v/v) Tween 20) and then blocked in 5% non-fat dry milk (NFDM) in TBST for 1 h at room temperature. Primary antibodies were incubated in TBST containing 1% NFDM overnight at 4 °C, whereas secondary antibodies were incubated in TBST containing 0.1% NFDM for 1 h at room temperature.
Proteins were visualized using enhanced chemiluminescence substrate ECL (Thermo Fisher Scientific, 32106) and imaged using a ChemiDoc XRS Bio-Rad Imager and Image Lab 6.0.0 software. Imaging was performed in two channels: chemiluminescence and colorimetry.
Quantitative PCR with reverse transcription (RT–qPCR)
RNA was extracted using an RNeasy Plus Mini kit (Qiagen, 74136) following the manufacturer’s instructions. The concentrations of RNA were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, 701-058112). The RNA was then reverse-transcribed using a Maxima First Strand cDNA Synthesis kit (Thermo Fisher Scientific, FERK1672), with two reverse transcription reactions being performed for each sample, each using 100 ng of RNA. The RT–qPCR assays were carried out using Fast SYBR Green master mix (Thermo Fisher Scientific, 4385617), and run on QuantStudio 6 and 7 Flex Real-Time PCR systems (Thermo Fisher Scientific). The cycling conditions involved an initial step at 95 °C for 15 min, followed by 40 two-step cycles (95 °C for 15 s; 60 °C for 60 s).
Luciferase ISG reporter assay
RAW-Lucia ISG cells are mouse macrophages containing an ISG-luciferase reporter. All cells were maintained following the manufacturer’s recommendations and incubated at 37 °C in a humidified atmosphere containing 5% CO2. RAW-Lucia ISG cells stably express an IFN-sensitive response element from the mouse ISG54 minimal promoter and five IFN-stimulated response elements (ISRE-ISG54) coupled to a synthetic coelenterazine-utilizing luciferase.
RAW-Lucia ISG cells were plated in 96-well plates (Corning, 3922) at 1 × 105 cells per well in 200 µl of the medium. Cells were allowed to adhere overnight before the medium was replaced with 180 µl of fresh medium. Target genes in pMMEC cells were knocked out by lentiviral infection. Medium from the pMMECs was centrifuged at 500 r.p.m. for 5 min before adding 20 µl to the RAW-Lucia ISG cells. After 24 h, 10 µl of medium from the RAW-Lucia ISG cells was added to a white-bottom 96-well plate, with 50 µl of QUANTI-Luc (Invivogen, rep-qlc1) detection reagent added immediately before luciferase detection. Luminescence was measured using a luminometer (SpectraMax i3x) with a 4 s start time and 0.1 s reading time.
Expression and purification of MRN complexes
The human MRN complex
MRE11–Flag/RAD50–6×His/NBS1–Flag was prepared by co-expression in Sf9 cells. In brief, the pFastBac1 plasmids containing genes for recombinant human MRE11–Flag, RAD50–6×HIS and NBS1–Flag40,41 were transformed into DH10Bac cells (Thermo Fisher Scientific, 10361012) to prepare individual bacmids. Individual bacmids were transfected into Sf9 cells to generate low titre P1 baculoviruses, which were subsequently used to prepare high titre P2 and P3 baculoviruses using the Bac to Bac system following the manufacturer’s recommendations (Invitrogen). For co-expression of the MRN complex, Sf9 cells (2.5 × 106 per ml) grown in Sf-900 III SFM (Thermo Fisher Scientific, 12659017) were co-infected with baculoviruses expressing MRE11–Flag, RAD50–6×HIS and NBS1–Flag, and the complex was expressed at 27 °C for 48 h. Cells were collected by centrifugation at 500g for 15 min at 4 °C and the cell pellet was rinsed with PBS and stored at −80 °C until purification.
Purification of the MRE11–Flag/RAD50–6×His/NBS1–Flag complex
The Sf9 cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM benzamidine and 0.2 mM PMSF) containing Roche complete EDTA-free protease inhibitor cocktail (Sigma-Aldrich, 11873580001) for 40 min with continuous stirring before centrifugation at 33,000g for 45 min at 4 °C. The supernatant was loaded by gravity flow onto a column packed with 3 ml of anti-Flag M2 affinity gel (Sigma-Aldrich, A2220) pre-equilibrated in wash buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10% glycerol and 1 mM benzamidine). Bound MRN complex was washed with wash buffer and eluted with wash buffer supplemented with 0.1 mg ml–1 purified 3×Flag peptide (Sigma-Aldrich, F4799). The eluted sample was diluted 2× with dilution buffer (25 mM Tris-HCl, pH 8.0, 10% glycerol and 10 mM β-mercaptoethanol) to decrease the NaCl concentration to 50 mM and immediately purified by anion-exchange chromatography with a Source Q resin (GE Healthcare, 17094705) using a gradient from 50 to 1,000 mM NaCl. Pooled fractions were further purified by gel filtration chromatography using a Superose 6 Increase 10/300 GL column (GE Healthcare, 29-0915-96) pre-equilibrated with 20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10% glycerol and 1 mM DTT. Purified MRN complex was concentrated using a Vivaspin 500 centrifugal concentrator (Vivascience) and stored at −80 °C. Sample purity was assessed by SDS–PAGE with Coomassie blue staining.
Purification of untagged MR and nuclease-dead MR complex
Sf9 cells were lysed as above. The supernatant was loaded onto a 4 ml strep-tactin XT (IBA Lifesciences, 2-4030-010) column, washed with wash buffer and then bound MR complex was eluted with wash buffer supplemented with 25 mM biotin. The eluted complexes were digested with TEV protease in dialysis buffer containing 25 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% glycerol and 10 mM β-mercaptoethanol overnight in at 4 °C. The digested MR complexes were further purified by Source Q and Superdex 200 increase columns (GE Healthcare, 28990944) as described above. Purified MR complexes were concentrated as above and stored at −80 °C. The NBS1–Flag protein was purified as described above for reconstitution with the untagged MR complex.
Mouse cGAS catalytic domain
The mouse cGAS catalytic domain containing residues 141–507 (mcGAS) was prepared as previously described4. The human cGAS catalytic domain (residues 157–522) was prepared as previously described44. The 6×His-Sumo tagged mouse cGAS catalytic domain (6×His-Sumo-mcGAS) used in the TR-FRET assay was expressed in BL21(DE3)pLysS cells. Cells were grown in 2XTY medium at 37 °C, and protein expression was induced through the addition of 0.2 mM IPTG when the OD600 reached 1.2 for 18 h at 16 °C. The 6×His-Sumo-mcGAS protein was purified using metal-affinity chromatography with Talon resin (Clontech, 635504), ion-exchange chromatography with Source S resin (GE Healthcare, 17094405) and size-exclusion chromatography with a Superdex 75 increase 10/300 GL column (GE healthcare), concentrated, supplemented with 20% glycerol and stored at −80 °C.
Fluorescent labelling of the mcGAS catalytic domain
The purified mcGAS was applied to a Zeba Spin desalting column (Thermo Fisher Scientific, 89878) for buffer exchange into labelling buffer (20 mM HEPES, pH 7.5, 150 mM NaCl and 1 mM TCEP) following the manufacturer’s recommendations. To facilitate in-gel detection, mcGAS was labelled with carboxyrhodamine. In brief, mcGAS was diluted to 0.5 mg ml–1 in labelling buffer and combined with one molar equivalent of 5-(and-6)-carboxyrhodamine 6G, succinimidyl ester (Thermo Fisher Scientific, C6157) and labelling was allowed to proceed for 2 h at 4 °C. Labelling was repeated with the addition of one more molar equivalent of fluorophore, followed by 2 h of incubation at 4 °C. Unreacted fluorophore was quenched through the addition of Tris-HCl, pH 8.0 to a final concentration of 10 mM. Carboxyrhodamine-labelled mcGAS (mcGAS-CR) was separated from quenched, unreacted fluorophore using a Zeba Spin desalting column as described above, and protein was quantitated using previously reported methods2.
Preparation of nucleosomes
Nucleosomes were reconstituted using recombinant human histones (H2A, H2B, H3.2 and H4) and 147 bp (1 bp symmetric linker DNA) or 185 bp (20 bp symmetric linker DNA) 601 nucleosome positioning sequence and purified by anion-exchange chromatography as previously described45. AP mutant nucleosomes were prepared using the H2A (E61A, E64S, N68A, D72S, N89A, D90A, E91S) mutants as previously reported45. For preparation of OG-NCPs, a H2A-H2B(A21C) dimer (2 mg ml–1) was incubated with Oregon Green 488 maleimide at a 1:1 ratio in labelling buffer (20 mM Tris-HCl, pH 7.5, 25 mM NaCl, 7 M guanidine HCl and 0.2 mM TCEP) for 2 h at 4 °C. Labelling was repeated with the addition of one more molar equivalent of fluorophore as above. The unfolded, labelled dimer was dialysed overnight at 4 °C in refolding buffer (10 mM HEPES, pH 7.5, 100 mM NaCl and 10 mM β-mercaptoethanol) and then purified by ion-exchange chromatography with a Source S resin (GE Healthcare). The labelled dimer was reconstituted into nucleosomes and purified as described above. The 147 bp nucleosomes were reconstituted with H2B(K125C)-biotin (biotin-NCP) or H2B(K125C)-Atto647N (647-NCP) for the nucleosome stacking assay. For this purpose, H2A-H2B(K125C) dimers were labelled with biotin-maleimide (Sigma-Aldrich, B1267) or Atto 647N maleimide (Sigma-Aldrich, 05316-1MG-F) as described above for Oregon Green 488 labelling.
TR-FRET assay
cGAS–MRN competition assays were performed as previously described25. In brief, 15 nM 6×His-Sumo-mcGAS, 2 nM biotin NCP, 4 nM LANCE Eu-W1024 streptavidin (PerkinElmer), 10 nM Ulight LANCE Ultra ULight-anti-6×His (PerkinElmer, TRF0134) and 0–2,000 nM of untagged MRN complex (mixture of 2 unlabelled MR:1 NBS1–Flag) or 0–5,000 nM unlabelled nucleosome were mixed in a total volume of 10 µl of TR-FRET 50 buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5% glycerol, 0.01% NP-40, 0.01% CHAPS, 5 mM DTT and 100 µg ml–1 BSA) in non-binding 384-well plates (Greiner Bio-One, 784904). Plates containing reaction mixture were shaken at 1,000 r.p.m. on a plate shaker for 1 min, centrifuged at 1,000 r.p.m. for 1 min and then incubated for 1 h at room temperature.
Nucleosome stacking by cGAS and MRN was tested by mixing 2 nM biotin-NCP, 50 nM 647-NCP, 2 nM LANCE Eu-W1024 streptavidin and 0–2,000 nM untagged MRN (mixture of 2 unlabelled MR:1 NBS1–Flag), 0–5,000 nM mcGAS or 0–5,000 nM hcGAS in TR-FRET 50 buffer.
For nucleosome stacking competition by MRN, the mixture containing 2 nM biotin-NCP, 50 nM 647-NCP, 2 nM LANCE Eu-W1024 streptavidin and 250 nM hcGAS was titrated with 0–2,000 nM MRN (mixture of 2 unlabelled MR:1 NBS1–Flag).
For all TR-FRET assays, FRET signals were measured after 1 h of incubation using 320 nm excitation and 615 nm/665 nm dual emission with a 100 µs delay between excitation and emission on an EnVision 2103 plate reader. All titrations were performed in triplicate. Competition data were fit to [inhibitor] versus response (three parameters) using GraphPad Prism v.7 software.
MRN–cGAS interaction by size exclusion
mcGAS-CR (5 µM) was incubated with 1 µM MRN complex in T50 buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl and 1 mM DTT) on ice for 20 min and then loaded onto a Superdex 200 Increase 10/300 column equilibrated with T50 buffer. The eluted fractions were separated by SDS–PAGE and fluorescence imaging was performed with a Typhoon FLA-9500 imager (GE Healthcare, excitation: 532 nm, emission: LPG low pass 575 nm), before Coomassie blue staining.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assays were performed by combining nucleosomes (50 or 100 nM final concentration) with serial dilutions of tagged MRN or MN complex and 200 nM mcGAS-CR in 10 µl of binding buffer (20 mM HEPES, pH 7.5, 50 mM NaCl, 5% sucrose and 1 mM DTT). Following 15–30 min equilibration on ice, samples were analysed by electrophoresis on 5% polyacrylamide gels run in 0.2× TBE at 150 V for 60 min at 4 °C. Gels were scanned to detect fluorescence signals of mcGAS-CR using a Typhoon FLA 9500 imager and then stained with ethidium bromide and imaged using a E-Gel Imager (Invitrogen).
In vitro assay for cGAS activity
Assays were performed as previously described4 with the following alterations. In vitro reactions (10 µl) reactions were assembled in PCR tubes with final concentrations of 0.5 µM or 1 µM purified mcGAS catalytic domain, 5 µM dsDNA (90 bp), 0.5 µM NCP, with varying (0–2 µM) MRN complex concentrations in reaction buffer (22 mM HEPES pH 7.5, 110 mM NaCl, 5.5 mM MgCl2, 5 mM DTT, 10 µM ZnCl2, 1.25 U of inorganic pyrophosphatase (Sigma-Aldrich, I1643) and 4% glycerol (final concentrations)). Reactions were equilibrated for 5 min at 37 °C and initiated with the addition of 2 mM (each) GTP/ATP mix containing about 12.5 nM [α-32P]ATP (PerkinElmer, NEG003X250UC) and incubated at 37 °C for 120 min.
Single-cell analysis
Single-cell RNA-seq library preparation
For 3′ single-cell RNA library construction, cells were processed using a 10x Genomics Chromium Controller and a Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v.3.1 (PN-1000121) following the manufacturer’s user guide (tinyurl.com/3we33fb6). In brief, 1:10 volume of 10% BSA was added to freshly sorted cells before centrifuging for 6 min at 800 r.c.f. in a 4 °C cold room. The supernatant was removed, and the cell pellet was resuspended in 25 µl of 1× PBS + 0.04% BSA. Aliquots of the sorted, concentrated cells were then stained with acridine orange and propidium iodide and assessed for viability and concentration using the LUNA-FL dual fluorescence cell counter (Logos Biosystems). Approximately 16,000 viable cells per sample were loaded onto a Chromium Chip G with a target recovery of 10,000 cells per sample for library preparation. Single cells, reverse transcription reagents and gel beads coated with barcoded oligonucleotides were encapsulated together in an oil droplet to produce gel beads in emulsion (GEMs). Reverse transcription was performed using a C1000 thermal cycler (Bio-Rad) to generate cDNA libraries tagged with a cell barcode and unique molecular index (UMI). GEMs were then disrupted and the cDNA libraries were purified using Dynabeads MyOne SILANE (Invitrogen) before 11 amplification cycles. Amplified libraries were purified using SPRIselect magnetic beads (Beckman Coulter) and quantified using an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent Technologies). Fragmentation, end repair, A-tailing and double-sided size selection using SPRIselect beads were then performed. Illumina-compatible adapters were ligated onto the size-selected cDNA fragments. Adapter-ligated cDNA was then purified using SPRIselect beads. Uniquely identifiable indexes were added during ten amplification cycles. The finalized sequencing libraries were purified using SPRIselect beads and visualized using an Agilent Bioanalyzer High Sensitivity DNA chip (Agilent Technologies).
Processing of scRNA-seq data
Data were imported into Seurat (v.3.1.2) using R (v.3.6.0). Cells with at least 5,000 UMIs and 1,000 genes detected and fewer than 10% mitochondrial contribution were retained for downstream analyses. Data were then normalized and scaled using scTransform, integrated using IntegrateData as previously described45, then clustered using Louvain–Jaccard clustering with multilevel refinement (resolution of 1.0). Unique marker genes were detected for each cluster using Presto. Differential expression analysis was performed on CPM-normalized counts using Seurat FindMarkers, and significantly enriched pathways among upregulated and downregulated genes were detected using gProfiler. Cell cycle scoring was then performed in Seurat to identify the different phases. We performed differential expression between sgControl and sgMRE11 in the G1 cells based on the nonparametric Wilcoxon rank sum test, following re-normalization as described above.
Orthotopic transplantation-based tumorigenesis assay
For mammary-fat-pad tumour assays, pMMECs were isolated from 8-week-old virgin female R26cas9/cas9, MycOE and Trp53fl/fl mice. After isolation, pMMECs were infected with 5 × 105 TU of indicated LentiCRISPR-Cre-V2-sgRNA lentiviruses. After a week, the pMMECs were collected by trypsinization, washed twice in PBS and counted. Cells were then resuspended (1 × 106 cells) in a 50:50 solution of PBS and Matrigel matrix (Corning, 356231). Each pMMEC preparation was injected into the left mammary fat pads of NOD.129S7(B6)-Rag1tm1Mom/J mice. Following the injections, the mice were monitored on a weekly basis for the development of tumours. After detection of tumour formation, the frequency of measurements was increased to three times a week.
The study adhered to humane end point criteria for tumour burden. Euthanasia was carried out when a single tumour reached a size of 2.0 cm in any direction. In cases when multiple tumours were present, the end point was established at 1.5 cm for any individual tumour. After reaching these end points, the mice were humanely euthanized in accordance with approved guidelines.
Z-RNA and Z-DNA staining in pMMECs
pMMECs were isolated from indicated 8-week-old virgin female R26cas9/cas9, MycOE and Trp53fl/fl mice, unless otherwise specified. Following isolation, pMMECs were infected with a multiplicity of infection of 3 using the indicated lentiviruses (sgControl, sgMre11, sgcGas or sgZbp1). Ten days after infection, cells were prepared for ICC to detect Z-RNA and Z-DNA. Cells were fixed with cold methanol for 10 min at 20 °C, then blocked with 3% BSA in PBS for 30 min. The cells were then incubated overnight at 4 °C with a Z-DNA antibody (Novous Biologicals, NB100-749) at a dilution of 1:500. The following day, cells were incubated with the secondary antibody (Alexa-594) for 1 h at room temperature. Cells that exhibited robust Z-RNA/Z-DNA signal aggregation were then counted. These signals manifested as either intense intranuclear clusters or as smaller aggregates dispersed around the perinuclear region.
Statistics and reproducibility
Statistical analyses are described in each figure legend. All data are plotted as averages, with error bars representing the s.e.m. unless stated otherwise. Data shown are representative of at least three independent biological replicates, unless otherwise indicated. Statistical analyses were performed using Prism (GraphPad Software). For all quantitative measurements, normal distribution was assumed, with t-tests performed unpaired and two-sided unless otherwise stated. For experiments combining several groups, an ordinary one-way ANOVA test was used. P > 0.05 was considered non-significant. Mouse tumorigenesis studies utilized publicly available sample size estimation calculators to attain at least 80% power to detect a 30% reduction in tumour latency using a two-tailed log-rank test. Sample sizes for in vitro experiments were determined empirically from previous experimental experience with similar assays and/or from sizes generally used in the field.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.