Low melting agarose

Single molecule analysis of resection tracks (SMART) was performed as described [28]. Briefly, cells were grown in the presence of 10 μm BrdU for < 24 h. Cultures were then irradiated (10 Gy) and harvested after 1 h. Cells were embedded in low‐melting agarose (Bio‐Rad, Hercules, CA, USA), followed by DNA extraction. DNA fibers were stretched on silanized coverslips, and immunofluorescence was carried out to detect BrdU (Table S2). Samples were observed with a Nikon NI‐E microscope, and images were taken and processed with the nis elements Nikon Software (Tokyo, Japan). For each experiment, at least 200 DNA fibers were analyzed, and the length of the fibers was measured with adobe photoshop cs4 (San Jose, CA, USA).

Samples (5 mL) were grown over night up A₆₀₀ = 1 × 10⁷, resuspended in 1 mL of broth, and then incubated for 4 h at 30°C. To label acidic organelles such as vacuoles, samples were resuspended in 250 μL 10 mmol/L HEPES buffer pH 7.4 containing 5% glucose with 100 μmol/L Cell Tracker Blue CMAC (Life Technologies), and incubated for 30 min at room temperature. Samples were spun for 3 min at 6700 g and the pellet resuspended in 1 mL of low melting agarose (Bio‐Rad) dissolved in the HEPES buffer. Aliquotes of 10 μL for each sample were seeded on glass bottom dishes (35 mm ø, ibidi) and covered with a 13 mm ø glass cover slip. Cells were observed with a laser scanning confocal microscope, TCS SP5 (Leica Microsystems, Mannheim) using a Plan Apo 63×(NA = 1.20) water‐immersion lens with optical pinhole at 1 AU. An Argon multiline laser operating at 488 nm (GFP), and a UV Diode laser at 405 nm (blue vacuole CMAC marker) were used as excitation sources. Confocal Z‐stacks were collected with 0.29 mm interval in a 5 mm total optical depth. Images for direct comparison were collected under the same parameters and representative images were chosen among 20 cells in multiple assays.

ER-AsiSI U2OS cells were prepared as previously described (47 (link)). Trypsinized cells were resuspended with 0.6% low-melting agarose (Bio-Rad) at a concentration of 1.2 × 10⁷ cells/ml. Fifty microliters of cell suspension was used to make an agar ball, which was incubated with ESP buffer (0.5 M EDTA, 2% N-lauroylsarcosine, 1 mg/ml proteinase K, 1 mM CaCl₂, pH 8.0) at 16°C for 20 h. The agar ball was treated with HS buffer (1.85 M NaCl, 0.15 M KCl, 5 mM MgCl₂, 2 mM EDTA, 4 mM Tris, 0.5% Triton X-100, pH 7.5) at 16°C for 20 h. Melted agar balls were incubated overnight with restriction enzyme (BsrGI or HindIII-HF; New England Biolabs). Real-time PCR was performed with restriction enzyme-treated or non-treated samples. The percentage of single-strand DNA (ssDNA) was calculated as described previously (47 (link)). Briefly, the Ct value was calculated by subtracting the Ct value of an untreated sample from that of a sample treated with the restriction enzyme. The ssDNA fraction was calculated (47 (link)) as .

Primary alveolar epithelial type II (AE2) cells were isolated from the mouse lung as previously described [29 (link)]. In brief, dispase (BD Bioscience, San Jose, CA) and low melting agarose (Bio-Rad; 1%, 0.5 ml) were sequentially instilled into the airways before extracting lungs en bloc and immediately placing tissues on ice to harden agarose. Next, lung tissues underwent mechanical digestion using the Gentle Macs tissue dissociator (Miltenyi Biotec). Purified preparations of AE2 cells were recovered using a two-step process that first removed CD45-expressing immune cells and then positively selected lung epithelial cells with an anti-Ep-CAM antibody [29 (link)].

SMART was performed as described18 (link). Briefly, U2OS cells downregulated for the indicated genes were grown in the presence of 10 μM BrdU for 24 h. Cultures were then irradiated (10 Gy) and harvested after 1 h. Cells were embedded in low-melting agarose (Bio-Rad), followed by DNA extraction. To stretch the DNA fibres, silanized coverslips (Genomic Vision) were dipped into the DNA solution for 15 min and pulled out at a constant speed (250 μm s⁻¹). Coverslips were baked for 2 h at 65 °C and incubated directly without denaturation with an anti-BrdU mouse monoclonal antibody (Supplementary Table 2). After washing with PBS, coverslips were incubated with the secondary antibody (Supplementary Table 3). Finally, coverslips were mounted with ProLong Gold Antifade Reagent (Molecular Probes) and stored at −20 °C. DNA fibres were observed with a Nikon NI-E microscope and a PLAN FLOUR 40 × 0.75 PHL DLL objective. The images were recorded and processed with the NIS ELEMENTS Nikon Software. For each experiment, at least 200 DNA fibres were analysed, and the length of DNA fibres was measured with Adobe Photoshop CS4 Extended version 11.0 (Adobe Systems Incorporated).

SMART was performed as described [29 ]. Briefly, cells were grown in the presence of 10 μM BrdU for 22–24 h. Cultures were then irradiated (10 Gy) and harvested after 1 h. Cells were embedded in low-melting agarose (Bio-Rad), followed by DNA extraction. DNA fibers were stretched on silanized coverslips, and immunofluorescence was carried out to detect BrdU (S2 and S3 Tables). Samples were observed with a Nikon NI-E microscope, and images taken and processed with the NIS ELEMENTS Nikon Software. For each experiment, at least 200 DNA fibers were analyzed, and the length of the fibers was measured with Adobe Photoshop CS4.

For live imaging, embryos were anesthetized in 0.016% tricaine (MS‐222; Sigma‐Aldrich) and embedded in 1.2% low melting agarose (Bio‐Rad, USA) in 35 mm glass‐bottom dishes (Eppendorf, Hamburg, Germany), and covered with 2‐mL fish medium containing tricaine. A Nikon SMZ18 epi‐fluorescence microscope was used for capturing whole‐embryo images. Z‐stack images and time‐lapse movies were recorded using a Nikon FV3000 confocal microscope (Nikon, Minato City, Tokyo, Japan) equipped with a ×30/1.05, WD 0.8 silicon oil objective. Time‐lapse files were processed using Imaris (Bitplane AG, Zurich, Switzerland) and Fiji (ImageJ; NIH, Bethesda, MD, USA; https://imagej.nih.gov/ij/) software to generate movies. Z‐stack images were processed into maximum intensity projections and channels merged using Fiji. Alizarin Red‐stained bone samples were imaged using a Nikon Eclipse 90i upright microscope equipped with NIS‐Elements version BR 3.0.