5 ethynyl 2 deoxyuridine (edu)

iPOND was performed as described by Sirbu et al. [45 (link)], with minor modifications. At least 1x10⁸ HeLa S3 cells per sample were pulsed with 10 μM EdU (Thermo Fisher Scientific) for the indicated times and either incubated with 10 μM thymidine (Merck) for 0–30 min before fixation (chase experiments) or fixated immediately (pulse experiments). For replication stress experiments thymidine containing medium was supplemented with 2 mM HU and/or 1 μM ABT-888. Click reaction to label EdU-containing DNA was performed using biotin-PEG3-azide (Jena Bioscience) for 90 min and cells were sonicated in a Bioruptor sonicator (Diogenode) to solubilize chromatin fragments. Biotin-linked fragments were precipitated overnight at 4°C using streptavidin-coupled magnetic beads (0.8 μm, Solulink). Chromatin bound proteins (“Capture”) were subjected to Western blot analysis using the following antibodies: polyclonal rabbit α-DEK K-877 (1:20,000; [21 (link)]), monoclonal mouse α-PCNA (1:9,000; PC10, Cell Signaling Technology), polyclonal rabbit α-H3 (1:150,000; ab1791, Abcam).

Asynchronously growing 2–3 × 10⁸ HeLa (MRL2 strain, a gift from O. Bensaude, IBENS) or 10⁹ GM06990 cells (Corriell) were labelled by adding 20 μM EdU (Jenabioscience) to growth media for 2 min. Adherent HeLa cells were washed with ice-cold 1 × phosphate-buffered saline (PBS) and harvested by scrubbing with a cell lifter. Non-adherent GM06990 cell suspensions were chilled in ice-cold water bath for 10 min and collected by centrifugation at 400 g for 10 min at 4 °C. As an alternative to EdU, 5-ethynyl-2′-deoxycytidine (EdC) was also used with identical results.

To identify S-phase cells, EdU (Jena Bioscience, Jena, Thuringia, Germany) was added into culture medium at 25 μM and incubated for 30 min. Incorporated EdU was visualized by a fluorescent dye through a Cu(I)-catalyzed [3 + 2] cycloaddition (click) reaction, by which terminal alkyne group of EdU is covalently attached with azide group of a fluorescent dye, as described previously with minor modifications [39 (link), 40 (link)]. Briefly, cells were fixed with 4% paraformaldehyde (PFA) for 15 min and permeabilized with 0.5% Triton X-100 for 3 min. After three times PBS washes, cells were incubated with the reaction mixture (2 μM picolyl azide Alexa-555 (Jena Bioscience), 20 mM Sodium ascorbate (FUJIFILM Wako Pure Chemical Corporation), 2 mM 2-(4-((bis((1-(tert-butyl)-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)acetic acid (Jena Bioscience), 1 mM CuSO4 (FUJIFILM Wako Pure Chemical Corporation) diluted in PBS) for 30 min at 37 °C. After three times PBS wash, cells were subjected to the immunostaining if necessary.

RFD profiling of DT40 cells by OK-seq was performed as described in (11 (link)) and (14 (link)). Briefly, asynchronously growing cells were labelled with 20 μM 5-ethynyl-2′-deoxyuridine (EdU, Jenabioscience) for 2 min. After DNA purification and heat-denaturation, Okazaki fragments were size-purified on sucrose gradients, labelled with biotin at EdU sites using click-chemistry, captured on magnetic beads coated with streptavidin and amplified by PCR. Sequencing was performed on HiSeq (Illumina) at the IB2C high-throughput sequencing platform (Gif-sur-Yvette, France). Aligned OK-seq data (BAM files) were imported in R (52 ) as GenomicAlignments, and reads were converted into GenomicRanges. RFD was computed using R as the difference between rightward- and leftward-fork coverage normalized by total coverage. Data were binned into nonoverlapping 10 kb windows. RFD profiles were exported into bigwig files using the export function from the rtracklayer package. The OK-seq experiment was performed three times. RFD profiles of the three biological replicates were highly similar, with Spearman's pairwise correlation coefficients computed in 10 kb windows ranging from 0.972 to 0.986. DMD and CCSER1 OK-seq-based RFD profiles for the three replicates are presented in Supplementary Figure S2.

Co‐cultures of T cells with N‐ and PE‐hAMSC were established in direct cell‐to‐cell contact. Around 10⁵ hAMSC were seeded in 96‐well plates (Nunc, Roskilde, Denmark) in 150 μl of UltraCulture medium (Lonza, Basel, CH, Switzerland) and irradiated (30 Gy) to block proliferation. The day after, mixed lymphocyte cultures (MLC) were obtained by culturing 10⁵ T lymphocytes and 10⁵ γ‐irradiated allogeneic PBMC in 100 μl of UltraCulture medium (Lonza), in the absence (controls) or presence of hAMSC. T‐cell proliferation was assessed by 5‐ethynyl‐2′deoxyuridine (EdU) incorporation as previously described 38. Briefly, 10 μM EdU (Life Technologies, Carlsbad, CA, USA) was added on day 5 and incubated for 16–18 hrs. Incorporated EdU was detected by the Cu‐catalysed alkyne‐azide cycloaddition (CuAAC or ‘click') reaction of the ethynyl group with 2.5 μM 3‐azido‐7‐hydroxycoumarin (Jena Biosciences, Jena, Germany), in buffer solution (100 mM Tris‐HCl pH 8.0, 10 mM L‐ascorbic acid, 2 mM CuSO₄) at RT for 30 min. The samples were acquired with a FACSAria (BD Biosciences) and analysed with FCS express v4.07 (DeNovo Software).