High-resolution mapping of mitotic DNA synthesis under conditions of replication stress in cultured cells

Summary

Cells experiencing DNA replication stress enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here we describe a protocol to identify at genome wide and at high resolution the genomic sites where MiDAS occurs in cells exposed to aphidicolin. We use EdU incorporation to label nascent DNA in mitotic cells, followed by isolation of the EdU-labeled DNA and next-generation sequencing.

For complete details on the use and execution of this protocol, please refer to Groelly et al. (2022)¹ and Macheret et al. (2020).²

Graphical abstract

Highlights

• •Mitotic DNA synthesis (MiDAS) is activated at discrete loci upon replication stress
• •Cell synchronization strategy to label sites of MiDAS with EdU
• •Isolation of EdU-labeled DNA followed by next-generation sequencing (NGS)
• •Bioinformatics tools for visualization and analysis of MiDAS-seq data

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Cells experiencing DNA replication stress enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here we describe a protocol to identify at genome wide and at high resolution the genomic sites where MiDAS occurs in cells exposed to aphidicolin. We use EdU incorporation to label nascent DNA in mitotic cells, followed by isolation of the EdU-labeled DNA and next-generation sequencing.

Before you begin

Optimize conditions for thymidine block

Timing: 1 week

During this step, we optimize conditions for cell synchronization at the G1/S transition (e.g., thymidine concentration and duration of treatment). To do this, we use flow cytometry analysis (FACS) of DNA content (Figure 1A), which will also enable us to determine the rate at which cells progress through S phase. All steps in the below protocol were performed with H1299 cells but can be adjusted for other cell lines.

• 1.Seed 1.6 × 10⁵ H1299 cells per well of a 6-well plate.
• 2.Place cells in the incubator for 24 h.
• 3.Treat cells with a range of thymidine concentration (e.g., 1–2 mM).
• 4.Place cells in the incubator for durations ranging from 16 to 24 h.
• 5.Collect cells using trypsinization.
• 6.Centrifuge cells at 300 × g for 5 min at 4°C.
• 7.Resuspend cell pellet in 0.5 mL of PBS and ensure single-cell solution by pipetting up and down several times.
• 8.Vortex at low speed and add ice-cold methanol, dropwise, to a final concentration of 90% (v/v).
• 9.Place fixed cells on ice for at least 1 h.
• 10.Centrifuge cells at 300 × g for 5 min at 4°C.
• 11.Resuspend cell pellet in 0.5 mL PBS containing 10 μL propidium iodide and 10 μL RNAseA. This is required for accurate cell cycle analysis as propidium iodide stains both DNA and RNA.
• 12.Incubate cells at room temperature for 1 h.

Pause point: Samples can be stored at +4°C for several days.

• 13.Perform FACS (i.e., plot histogram of propidium iodide vs cell count).

Note: The ideal condition for thymidine block should give a sharp G1 peak (2C DNA content) with minimal contamination from other phases of the cell cycle (Figure 1A, 0 h after release). Once this condition is identified, use this thymidine concentration and duration of treatment to release cells into S phase as described below and to monitor their progression.

• 14.Block cells with thymidine, wash them three times with 1 mL PBS and release them in 1.5 mL pre-warmed culture medium. Collect cells every hour after release from thymidine as described in steps 5–7 and prepare them for FACS analyses as in steps 8–13 (Figure 1A). Troubleshooting 1.

Figure 1.

Flow cytometry analysis of H1299 cells synchronized for MiDAS-seq

(A) Flow cytometry analysis (propidium iodide) of H1299 cells synchronized at the G1/S transition with 1.5 μM thymidine for 16 h and released for the indicated duration.

(B) Flow cytometry (propidium iodide and phospho-Histone H3) of H1299 cells collected 12 h after release from thymidine block and treated as indicated.

(C) Flow cytometry analysis (propidium iodide) of H1299 cells synchronized at the G1/S transition with 1.5 μM thymidine for 16 h and released in the presence of 6 μM RO-3306 and 0.4 or 0.2 μM aphidicolin for the indicated duration.

(D) Flow cytometry analysis (propidium iodide and phospho-Histone H3) of H1299 cells synchronized at the G1/S transition with 1.5 μM thymidine for 16 h, released in the presence of 6 μM RO-3306 and 0.2 μM aphidicolin for 17.5 or 20.5 h and released into mitosis for 1.5 h. Hydroxyurea was added during the last 3 h.

(E) Experimental timeline for MiDAS-seq in H1299 cells.

Optimize conditions for reversible G2 arrest

Timing: 1 week

During this step, we determine the optimal concentration of CDK1 inhibitor (RO-3306) required to effectively arrest cells released from thymidine block in G2. To do this, we use FACS analyses of DNA content combined with immunofluorescent staining for phosphorylated Histone H3 (Ser10)¹ (Figure 1B), which is a marker for cells that entered mitosis.

• 15.Seed 1.6 × 10⁵ H1299 cells per well of a 6-well plate.
• 16.Place cells in the incubator for 24 h.
• 17.Synchronize cells at the G1/S transition as in the previous protocol.
• 18.Wash cells three times with 1 mL PBS.
• 19.Release cells in 1.5 mL pre-warmed culture medium.
• 20.Treat cells with a range of RO-3306 concentration (e.g., 4–10 μM).

Note: Include a non-treated or solvent-treated sample.

• 21.Place cells in the incubator for several hours, until most of the untreated cells have entered mitosis, based on the FACS analyses established in the previous protocol (e.g., ≥ 9 h based on Figure 1A).
• 22.Collect cells with trypsin.
• 23.Centrifuge at 300 × g for 5 min at 4°C.
• 24.Resuspend cells in 0.5 mL of PBS and ensure single-cell solution by pipetting up and down several times.
• 25.Vortex the cell solution at low speed and add ice-cold methanol, dropwise, to a final concentration of 90% (v/v).
• 26.Place fixed cells on ice for at least 1 h.
• 27.Centrifuge at 350 × g for 5 min.
• 28.Resuspend in 1 mL of 1× Click-iT saponin-based permeabilization and wash reagent.
• 29.Incubate at room temperature for 20 min.
• 30.Centrifuge at 350 × g for 5 min.
• 31.Remove supernatant and wash once with 1 mL 2% FBS in PBS.
• 32.Centrifuge cells at 350 × g for 5 min.
• 33.Resuspend cell pellet in 100 μL primary antibody solution: 2 μL mouse anti-phosphorylated-Histone H3 (Ser10) antibody diluted in 2% FBS in PBS.
• 34.Incubate cells for 90 min at room temperature, pipetting up and down every 15–30 min.
• 35.Add 1 mL 2% FBS in PBS and spin at 350 × g for 5 min.
• 36.Resuspend cell pellet in 100 μL secondary antibody solution: 0.5 μL goat anti-mouse Alexa Fluor 488 diluted in 2% FBS in PBS.
• 37.Incubate cells for 60 min at room temperature in the dark, pipetting up and down every 15–30 min.
• 38.Wash cells in 1 mL PBS.
• 39.Resuspend cell pellet in 0.5 mL PBS containing 10 μL propidium iodide and 10 μL RNAseA.
• 40.Incubate for 1 h at room temperature.

Pause point: Samples can be stored at +4°C for several days.

Alternatives: Staining of phosphorylated Histone H3 (Ser10) can also be done using antibodies specific for FACS (which are conjugated to fluorophores).

• 41.Perform FACS analyses of DNA content and phosphorylated Histone H3 (Ser10) (i.e., plot propidium iodide vs phosphorylated Histone H3 (Ser10) signal, Figure 1B).

Note: Select the lowest concentration of RO-3306 which prevents mitotic entry (determined by lack of phosphorylated Histone H3 (Ser10)-positive cell population) at the selected time point after G1/S release. Troubleshooting 2.

Optimize conditions for aphidicolin-induced replication slowdown

Timing: 1 week

During this step, we optimize aphidicolin concentrations that effectively reduce the rate of cell progression through S-phase. Because aphidicolin is an inhibitor of the replicative DNA polymerases alpha and delta, it acts by slowing down replication fork progression. We use FACS analyses of DNA content (Figure 1C) to monitor the increase in the percentage of S-phase cells, which is indicative of replication slowdown.

• 42.Seed 1.6 × 10⁵ H1299 cells per well of a 6-well plate.
• 43.Place cells in the incubator for 24 h.
• 44.Synchronize cells at the G1/S transition in the previous protocol.
• 45.Wash cells three times with 1 mL PBS.
• 46.Release cells in 1.5 mL pre-warmed culture medium containing RO-3306 (using the previously optimized concentration for G2 arrest) and a range of aphidicolin concentrations (e.g., 0.1–0.4 μM).
• 47.Collect cells every 2 h, typically from 8 h to 24 h after release from thymidine block, and perform FACS analyses for DNA content as described in the previous protocol to monitor mitotic entry (Figure 1C). Troubleshooting 3.

Determine the timing for collecting mitotic cells

Timing: 1–2 weeks

During this step, we determine the timing of the release from RO-3306 block and the optimal duration of release that will ensure that cells progressed into early mitosis (Figure 1D).

• 48.Seed 1.6 × 10⁵ H1299 cells per well of a 6-well plate.
• 49.Place cells in the incubator for 24 h.
• 50.Synchronize cells at the G1/S transition and release in RO-3306 and aphidicolin-containing medium using conditions identified using previous protocols.
• 51.Place cells in the incubator for several hours until cells reach G2.

Note: Test several conditions. For instance, cells treated with 0.2 μM aphidicolin seem to reach G2 between 16 and 20 h after release from thymidine.

• 52.Treat cells with 2 mM hydroxyurea for 90 min.
• 53.Wash cells three times with 1.5 mL pre-warmed culture medium supplemented 2 mM hydroxyurea.

CRITICAL: The total duration of the washes should not exceed 5 min.

• 54.Release cells in 1.5 mL pre-warmed culture medium supplemented with 2 mM hydroxyurea and 100 ng/mL nocodazole.

Alternatives: Cells can also be arrested in mitosis using colcemid (e.g., KaryoMax).

• 55.Place cells in the incubator for 1–2 h.
• 56.Wash cells and perform FACS analyses of DNA content and phosphorylated Histone H3 (Ser10) (i.e., plot propidium iodide vs phosphorylated Histone H3 (Ser10) signal, Figure 1D).

Note: The presence of mitotic cells can first be assessed under the light microscope since mitotic cells tend to round up.

Note: Prolonged G2 arrest can be toxic to the cells (Figure 1D, 19 h versus 22 h after release from thymidine). Troubleshooting 2, 3 and 4.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Histone H3 Phospho S10 primary antibody (mouse)	Cell Signaling	Cat# 9701; RRID: AB_331535
Alexa Fluor 488 goat anti-mouse	Thermo Fisher	Cat# A10667; RRID: AB_2534057
Chemicals, peptides, and recombinant proteins
Dulbecco’s modified Eagle’s medium (DMEM)	Sigma-Aldrich	Cat# D5796
Tet system approved fetal bovine serum	Takara Bio	Cat# 631106
Trypsin-EDTA (0.05% (wt/vol))	Thermo Fisher	Cat# 25300054
Doxycycline	Sigma-Aldrich	Cat# D9891
Thymidine	Sigma-Aldrich	Cat# T1895
RO-3306	Sigma-Aldrich	Cat# SML0569
Hydroxyurea	Sigma-Aldrich	Cat# H8627
Nocodazole	Sigma-Aldrich	Cat# M1404
EdU	Thermo Fisher	Cat# C10340
Aphidicolin	Sigma-Aldrich	Cat# A0781
Azide-PEG(3+3)-S-S-biotin	Jena Biosciences	Cat# CLK-A2112-10
Dynabeads MyOne streptavidin C1	Thermo Fisher	Cat# 65001
2-β-mercaptoethanol	Sigma-Aldrich	Cat# M6250
Propidium iodide solution (1 mg/mL)	Sigma-Aldrich	Cat# P4864
PureLink RNase A (20 mg/mL)	Thermo Fisher	Cat# 12091021
Triton-X-100	Sigma-Aldrich	Cat# X100-500ML
PBS	Thermo Fisher	Cat# BR0014G
CuSO4	Sigma-Aldrich	Cat# 451657
Sodium-L-ascobate	Sigma-Aldrich	Cat# A7631
1 M Tris, pH 8	Santa Cruz Biotechnology	Cat# sc-296649
EDTA	Lonza	Cat# 51201
NaCl	Sigma-Aldrich	Cat# S3014
Tween-20	Sigma-Aldrich	Cat# P7949
1× Click-iT saponin-based permeabilization and wash reagent (from Click-iT EdU Flow Cytometry Assay Kit)	Thermo Fisher	Cat# C10424
Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	Cat# L3771
Proteinase K	Roche	Cat# 03115844001
Ethanol	Sigma-Aldrich	Cat# 34860
Methanol	Sigma-Aldrich	Cat# 51976
Phenol/chloroform/isoamyl alcohol (25:24:1)	Sigma-Aldrich	Cat# P3803
Chloroform/isoamyl alcohol (24:1)	Sigma-Aldrich	Cat# C0549
Glycogen (5 mg/mL)	Thermo Fisher	Cat# AM9510
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	Cat# D2650
Critical commercial assays
Qubit dsDNA BR Assay Kit	Thermo Fisher	Cat# Q32850
Qubit dsDNA HS Assay Kit	Thermo Fisher	Cat# Q32851
TruSeq ChIP Library Preparation Kit	Illumina	Cat# IP-202-1012
Deposited data
MiDAS-seq data	Groelly et al.1	GEO: GSE196163
Experimental models: Cell lines
H1299 +shBRCA2DOX cells (male origin)	Zimmer et al.4	N/A
Software and algorithms
DeepTools	Ramírez et al.5	https://deeptools.readthedocs.io/en/develop/
IGV-Web app version 1.7.0	Robinson et al.6	https://software.broadinstitute.org/software/ igv/
EdU-seq processing and plotting	Macheret and Halazonetis3	N/A
FlowJo	BD Biosciences	https://www.flowjo.com/

Materials and equipment

• •Thymidine: 200 mM in ddH₂O.

Store at −20°C for several months.

• •Aphidicolin: 1 mM in DMSO.

Store at −20°C for several months.

• •RO-3306: 10 mM in DMSO.

Store at −20°C for several months.

• •Hydroxyurea: 1 M in ddH₂O.

Store at −20°C for several months.

• •Nocodazole: 1 mg/mL in DMSO.

Store at −20°C for several months.

• •EdU: 10 mM in DMSO.

Store at −20°C for several months.

• •Permeabilization buffer: 0.2% (v/v) Tritom-X-100 in PBS.

Store at room temperature for several months.

• •Cleavable biotin-azide conjugate (working solution): 10 mM in DMSO.

Store at −20°C for several months.

• •CuSO₄(working solution): 100 mM in ddH₂O.

Store at +4°C for several months.

• •Sodium-L-ascobate (working solution): 1 M in ddH₂O.

Store at −20°C for several months.

Note: The recipes are given to prepare 1 mL of solution. Adjust the volumes depending on the number of samples and of cells per sample.

Biotin-azide click reaction cocktail
Reagent	Final concentration	Amount
Tris, pH 8 (100 mM)	86 mM	855 μL
CuSO4 (100 mM)	4 mM	40 μL
Sodium-L-ascobate (1 M)	100 mM	100 μL
Cleavable biotin azide (10 mM)	50 μM	5 μL
Total	N/A	1 mL

Prepare before use.

CRITICAL: CuSO₄ is harmful if swallowed and causes skin irritation. Wear appropriate eye protections and gloves.

Lysis buffer
Reagent	Final concentration	Amount
Tris, pH 8 (1 M)	10 mM	10 μL
EDTA (0.5 M)	10 mM	20 μL
SDS (20%)	0.5% (v/v)	25 μL
Proteinase K (20 mg/mL)	0.2 mg/mL	10 μL
ddH2O	N/A	935 μL
Total	N/A	1 mL

Prepare before use.

CRITICAL: SDS is harmful if swallowed or inhaled and causes skin irritation. Wear appropriate eye protections.

2× Bind and Wash buffer (2× BW buffer)
Reagent	Final concentration	Amount
Tris-HCl pH 7.5 (1 M)	10 mM	10 μL
EDTA (0.5 M)	1 mM	2 μL
NaCl (5 M)	2 M	400 μL
Tween-20	1% (v/v)	10 μL
ddH2O	N/A	578 μL
Total	N/A	1 mL

Prepare before use.

1× Bind and Wash buffer (1× BW buffer)
Reagent	Final concentration	Amount
2× BW buffer	N/A	500 μL
ddH2O	N/A	500 μL
Total	N/A	1 mL

Prepare before use.

TE buffer
Reagent	Final concentration	Amount
Tris pH 8 (1 M)	10 mM	10 μL
EDTA (0.5 M)	1 mM	2 μL
ddH2O	N/A	988 μL
Total	N/A	1 mL

Store at room temperature for several months.

Elution buffer
Reagent	Final concentration	Amount
Tris pH 8 (1 M)	10 mM	10 μL
2-β-mercaptoethanol	2% (v/v)	20 μL
ddH2O	N/A	970 μL
Total	N/A	1 mL

Prepare before use.

CRITICAL: 2-β-mercaptoethanol is toxic if swallowed, inhaled or in contact with skin. Wear appropriate eye protections and gloves, and handle in a fume hood.

Step-by-step method details

Cell culture for MiDAS-seq

Timing: 1–2 weeks

During this step, previously optimized conditions are used to synchronize cells at the G1/S transition, release cells into S-phase in the presence of RO-3306 and aphidicolin, and subsequently release them again into mitosis in the presence of EdU (Figure 1E). Mitotic cells thus prepared have incorporated EdU at MiDAS sites and are collected by mitotic shake-off.

Note: The incorporation of EdU in mitotic cells can be confirmed by preparing cells for detection of mitotic EdU foci by fluorescent microscopy.^1,2,7

• 1.Seed 1.25 × 10⁶ H1299 cells per T175 flask (15 flasks per condition).

Note: The number of flasks to use depends on the number of mitotic cells recovered in previous optimization sections. We recommend aiming for at least 10⁶ mitotic cells per experiment.

• 2.Place cells in the incubator for 48 h.
• 3.Treat cells with 1.5 mM thymidine.
• 4.Place cells in the incubator for 16 h.
• 5.Wash cells with three times with 10 mL PBS.
• 6.Add 12 mL pre-warmed culture medium per flask supplemented with 6 μM RO-3306 and 0.2 μM aphidicolin.
• 7.Place cells in the incubator for 10.5 h.

CRITICAL: The duration and dose of aphidicolin treatment should delay S phase progression without preventing mitotic entry. The duration and dose of RO-3306 treatment should ensure that the cells are reversibly arrested in G2.

Note: Here, low-dose aphidicolin treatment is used to slow down replication fork progression. Cells carrying certain genetic alterations (e.g., BRCA2 gene inactivation) also exhibit slow replication fork progression. The protocols above should be optimised accordingly for cells of each genotype.

• 8.Treat cells with 2 mM hydroxyurea.
• 9.Place cells in the incubator for 1.5 h.
• 10.Wash cells three times with 5–10 mL pre-warmed culture medium supplemented 2 mM hydroxyurea.

CRITICAL: The total duration of the washes should not exceed 5 min.

• 11.Add 12 mL pre-warmed culture medium per flask supplemented with 2 mM hydroxyurea, 100 ng/mL nocodazole and 10 μM EdU.
• 12.Place cells in the incubator for 1.5 h.
• 13.Perform a mitotic shake-off (e.g., gently tap the flask against a hard surface or with the hand) to detach the loosely attached mitotic cells, without lifting interphase cells. Troubleshooting 5.

Note: Adapt the strength of the mitotic shake-off to each cell line. For example, H1299 cells reaching mitosis tend to stick to the flask, therefore a stronger shake-off is required. If many mitotic cells still remain attached, then the number of flasks should be increased.

• 14.Collect the medium containing floating mitotic cells in 50-mL tubes.
• 15.Centrifuge cells at 300 × g for 5 min.
• 16.Wash cells with PBS and transfer to one 15-mL tube per condition.
• 17.Centrifuge cells at 300 × g for 5 min.
• 18.Resuspend cell pellets in 1 mL PBS. Make sure to obtain a single-cell solution by pipetting up and down several times.
• 19.Vortex cell solution at low speed and add ice-cold methanol, dropwise, to a final concentration of 90% (v/v).
• 20.Place fixed cells on ice for at least 1 h.

Pause point: The cells can be stored at −20°C up to several months.

Cell permeabilization and the ‘click’ reaction

Timing: 3 h

During this step, a cleavable biotin-azide is crosslinked to the EdU-labeled DNA using ‘click’ chemistry.

Note: The protocol below is adapted from Macheret and Halazonetis.⁸

• 21.Centrifuge cells at 350 × g for 10 min at 4°C.
• 22.Wash cell pellets with 3 mL of ice-cold PBS.
• 23.Centrifuge cells at 350 × g for 10 min at 4°C.
• 24.Resuspend cell pellets in 1 mL permeabilization buffer per 3 × 10⁶ cells.
• 25.Incubate the cell suspension at room temperature for 30 min.
• 26.Centrifuge cells at 350 × g for 10 min at 4°C.
• 27.Wash cells with 3 mL of PBS.
• 28.Centrifuge cells at 350 × g for 10 min at 4°C.

Note: Ensure that pipet tips are not contaminated with 2-β-mercaptoethanol or dithiothreitol (DTT), as this would cleave the biotin-azide and thus prevent binding of the EdU-labelled DNA to the streptavidin beads.

• 29.Prepare the biotin-azide ‘click’ reaction cocktail according to the table above (see materials and equipment). Prepare 250 μL per 3 × 10⁶ cells.
• 30.Resuspend cell pellets in 250 μL of biotin-azide click reaction cocktail per 3 × 10⁶ cells.
• 31.Incubate cell suspensions for 30 min at room temperature and in the dark.
• 32.Centrifuge cells at 350 × g for 10 min at 4°C.
• 33.Wash cells in 3 mL PBS.

Isolation of EdU-labeled DNA

Timing: 3–5 days

During this step, DNA is purified and fragmented using sonication. Next, the EdU-labeled DNA is isolated using streptavidin beads that bind to the EdU biotinylated in the previous protocol. Finally, EdU-labeled DNA is prepared for next-generation sequencing.

• 34.Centrifuge cells at 350 × g for 10 min at 4°C.
• 35.Resuspend cells in 400 μL lysis buffer.
• 36.Transfer cells to a 1.5 mL tube.
• 37.Incubate samples in a water bath at 50°C for at least 3 h.

Pause point: The samples can be incubated overnight at 50°C.

• 38.Add 1 volume (400 μL) of phenol/chloroform/isoamyl alcohol (25:24:1). Mix by flicking and transfer to a new 1.5 mL tube.
• 39.Incubate for 5 min at room temperature.
• 40.Spin in a microcentrifuge at maximum speed for 5 min, at room temperature.
• 41.Recover the aqueous phase and transfer to a new 1.5-mL tube.
• 42.Add 1 volume (400 μL) of chloroform/isoamyl alcohol. Mix by inverting.
• 43.Spin in a microcentrifuge at maximum speed for 5 min at room temperature.
• 44.Recover the aqueous phase (taking care not to touch the interphase) and transfer to a new 1.5-mL tube.
• 45.Add NaCl to a final concentration of 0.2 M (16 μL of 5 M NaCl). Mix well.
• 46.Add 1 μL of glycogen as carrier.
• 47.Precipitate the DNA by adding 2 volumes (832 μL) of ice-cold ethanol.
• 48.Incubate for 2 h at −20°C.

Pause point: The samples can be kept at −20°C for several months.

• 49.Centrifuge at maximum speed for 30 min at 4°C.
• 50.Aspirate and discard the supernatants. Be careful not to disrupt the pellets.
• 51.Wash with 70% ice-cold ethanol.
• 52.Spin in a micro-centrifuge at maximum speed for 15 min at 4°C.
• 53.Aspirate and discard the supernatants. Be careful not to disrupt the pellets.
• 54.Let the pellet dry for 5 min at room temperature.
• 55.Add 55 μL TE buffer.
• 56.Let the DNA resuspend at room temperature for several hours.

Pause point: The samples can be left at room temperature overnight or stored at −20°C for up to several months.

• 57.Measure the DNA concentration using a Qubit fluorometer with dsDNA BR assay kit.
• 58.Transfer 5–15 μg of DNA to a new 1.5-mL tube. Adjust the volume to 105 μL with water.

Note: If the starting material is lower, proceed with as much DNA as possible.

Optional: Retrieve 5 ng DNA to be used as a chromatin input sample to generate baseline for the sequencing profiles.

• 59.Use a bioruptor to sonicate the DNA into fragments of 100–500 bp.

CRITICAL: The bioruptor settings should be optimized to obtain DNA fragments of 250 bp in length (range 100–500 bp). The size of the DNA fragments can be checked on an agarose gel. For instance, chromatin was sheared for 6 sonication cycle (15 s ON /90 s OFF) using a bioruptor PICO from Diagenode. Note that efficiency of the sonication depends on the type of tube used.

• 60.Run 2 μL of sonicated DNA on a 1% agarose gel and assess the length of DNA fragment using gel imaging system.
• 61.Resuspend the Dynabeads MyOne Streptadvin C1 beads in the vial by vortexing.
• 62.Transfer 50 μL of bead suspension in one 1.5-mL tube per sample.
• 63.Place the tube on the magnet for 1–2 min. Carefully aspirate and discard the preservative solution while the tube is on the magnet. It is essential not to let the beads dry.
• 64.Remove the tube from the magnet and wash the beads with 50 μL of 1×BW buffer.
• 65.Resuspend the beads by vortexing at low speed and perform a short spin (1,000 × g, 20°C, 5 s).
• 66.Place the tube on the magnet for 1–2 min. Carefully aspirate and discard the supernatants while the tube is on the magnet.
• 67.Repeat the washing steps two more times.
• 68.Resuspend the beads in 100 μL 2×BW buffer.
• 69.Transfer 100 μL of beads to a new 1.5-mL tube. Add 100 μL of sonicated DNA.
• 70.Incubate the samples for 15 min at room temperature on a rotating wheel to allow the binding of the EdU-labeled DNA.
• 71.Place the tube on the magnet for 1–2 min. Carefully aspirate and discard the supernatants while the tube is on the magnet.
• 72.Remove the tube from the magnet and wash the beads with 200 μL of 1×BW buffer.
• 73.Resuspend the beads by vortexing at low speed and perform a short spin (1,000 × g, 20°C, 5 s).
• 74.Place the tube on the magnet for 1–2 min. Carefully aspirate and discard the supernatants while the tube is on the magnet.
• 75.Repeat the washing steps two more times.
• 76.Remove the tube from the magnet and wash the beads with 200 μL of TE.
• 77.Resuspend the beads by vortexing at low speed and perform a short spin (1,000 × g, 20°C, 5 s).
• 78.Place the tube on the magnet for 1–2 min. Carefully aspirate and discard the supernatants while the tube is on the magnet.
• 79.Remove the tube from the magnet and add 55 μL of elution solution (2-β-mercaptoethanol).
• 80.Resuspend the beads by pipetting.
• 81.Incubate for 1 h at room temperature.
• 82.Place the samples on the magnet for 1–2 min. Recover the supernatants, which contains the eluted EdU-labeled DNA, while the tube is on the magnet. Transfer to a new 1.5-mL tube.
• 83.Measure the DNA concentration with a Qubit fluorometer with dsDNA BR assay kit using 5 μL of each sample.

CRITICAL: At this point, you may not be able to quantify the chipped DNA. However, you can still process to the library preparation and use as much DNA as you have.

Pause point: The samples can be stored at −20°C for days.

• 84.Use 5 ng of eluted DNA to prepare sequencing libraries according to the manufacturer’s instructions (TruSeq ChIP Library Preparation Kit, Illumina).
• 85.Perform high-throughput 100-bp single-end sequencing using an Illumina Hi-Seq 4000 sequencer, aiming for at least 20 × 10⁶ reads per sample.

Optional: Perform the library preparation and high-throughput sequencing steps for the chromatin input samples as well.

Expected outcomes

This protocol should generate files containing sequencing reads of regions enriched for EdU incorporation, which correspond to MiDAS events. The raw sequencing files can be analyzed as described below in order to visualize MiDAS peaks at common fragile sites. Troubleshooting 5.

Note: Which genomic regions undergo MiDAS upon aphidicolin treatment is cell line specific.

Quantification and statistical analysis

• 1.Align raw sequencing file to a reference genome (e.g., GRCh37/hg19) using the Burrows-Wheeler Aligner software:

bwa mem reference_genome_hg19.fastq myMiDASseq_experiment.fastq.gz > myMiDASseq_experiment.sam

• 2.The MiDAS-seq signal across 10-kb genomic bins can be calculated as sigma-values and plotted using previously described scripts.³ The sigma-value can also be used to identify MiDAS sites (peaks) by searching local maxima (Figure 2).¹

• 3.
  Alternatively, the MiDAS-seq signal across 10-kb genomic bins can be calculated using deepTools⁵ and visualized on the IGV-Web app version 1.7.0 (Figure 2).⁶

  • a.
    Convert an aligned SAM file into a BAM file (more details on samtools (http://quinlanlab.org/tutorials/samtools/samtools.html):

    samtools view -S -b myMiDASseq_experiment.sam > myMiDASseq_experiment.bam

  samtools view -S -b myMiDASseq_experiment.sam > myMiDASseq_experiment.bam

  .

  • b.
    Sort and index a BAM file:

    samtools sort myMiDASseq_experiment.bam -o myMiDASseq_experiment.bam

    samtools index myMiDASseq_experiment.sorted.bam

  samtools sort myMiDASseq_experiment.bam -o myMiDASseq_experiment.bam

  samtools index myMiDASseq_experiment.sorted.bam

  • c.
    Convert a BAM file into a BigWig (more details on deepTools (https://deeptools.readthedocs.io/en/latest/content/tools/bamCoverage.html):

    bamCoverage --bam myMiDASseq_experiment.sorted.bam -o myMiDASseq_experiment.bw \ --binSize 10000

  bamCoverage --bam myMiDASseq_experiment.sorted.bam -o myMiDASseq_experiment.bw \ --binSize 10000

  • d.
    Open the IGV-Web app (https://igv.org/app/) and navigate under Tracks > Local file to open BigWig file.

Figure 2.

MiDAS-seq signal at representative loci

MiDAS-seq profiles at representative genomic regions for H1299 cells treated with 0.2 μM aphidicolin.

(A) MiDAS-seq data were processed and plotted using previously described scripts³ (see quantification and statistical analysis, step 2). RT, replication timing (green, mid; yellow, late); Ge, genes (green, forward direction of transcription; red, reverse direction of transcription); IGe, intergenic regions.

(B) MiDAS-seq data were processed using deepTools and plotted using the IGV-Web app (see quantification and statistical analysis, step 3).

Limitations

Because this protocol relies on the enrichment of mitotic cell by mitotic shake off, it is only suitable for adherent cell lines. The strength of the mitotic shake off should also be optimized to maximize the number of mitotic cells collected while avoiding detaching interphase cells.

Troubleshooting

Problem 1

Cells don’t progress synchronously through S phase.

Potential solution

• •Adjust thymidine concentration and the duration of the block.
• •Ensure washes are quick and cells are placed back in the incubator rapidly.
• •Explore other synchronization methods (e.g., contact inhibition, serum starvation).

Problem 2

Cells don’t enter mitosis after release from RO-3306 arrest.

Potential solution

• •Monitor S-phase progression in cells treated with RO-3306 since the treatment can delay S phase progression.
• •Decrease RO-3306 concentration.
• •Alter the duration of the RO-3306 treatment since prolonged G2 arrest may be toxic.

Problem 3

Aphidicolin treatment is toxic or prevents mitotic entry.

Potential solution

• •Decrease aphidicolin concentration.
• •Alter the duration of the RO-3306 and/or aphidicolin treatment since prolonged treatment may be toxic.

Problem 4

Low number of mitotic cells following mitotic shake-off (step 13).

Potential solution

• •Decrease aphidicolin concentration to reduce replication stress and increase the fraction of cells entering mitosis.
• •Decrease RO-3306 concentration.
• •Alter the duration of the RO-3306 and/or aphidicolin treatment.
• •Alter the strength of the mitotic shake-off to collect.
• •Use a cell line which is strongly adherent in interphase and easily detaches in mitosis (e.g., HeLa cells).

Problem 5

No MiDAS peaks visible in sequencing files.

Potential solution

• •Increase the number of mitotic cells collected.
• •Adjust the aphidicolin concentration to increase the replication stress levels.
• •Perform genome-wide analysis, as loci prone to MiDAS upon replication stress may vary between cell lines.
• •Perform manual analysis rather than automated peak-calling since parameters for peak-calling algorithms need to be carefully optimized.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Madalena Tarsounas (madalena.tarsounas@oncology.ox.ac.uk).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The MiDAS-seq data used in the paper are available under the GEO accession number: GSE196163. The codes used to analyze MiDAS-seq data are available from previous publications.^1,3