In vivo psoralen cross-linking, isolation of genomic DNA from mammalian cells, enrichment of replication intermediates, and analysis of data was performed as previously described (Neelsen et al., 2014 (link); Thangavel et al., 2015 (link)). Briefly, 5–10 × 106 Mouse Embryonic Fibroblasts (Rosa+/ERCRE Atm+/C) cells were harvested and genomic DNA was cross-linked by three rounds of incubation in 10 µg/ml 4,5′,8-trimethylpsoralen (Sigma-Aldrich) and 3 min of irradiation with 366 nm UV light on a precooled metal block. Cells were lysed and cellular proteins were digested with 2 mg proteinase K (Life technologies) in 5 ml digestion buffer. DNA was purified by isopropanol precipitation, restriction digested with PvuII HF for 4 hr at 37°C, and replication intermediates were enriched using benzolylated naphthoylated DEAE-cellulose (Sigma–Aldrich) in 3 ml BioRad poly-prep chromatography columns. Samples were prepared by spreading DNA on carbon-coated grids in the presence of benzyl-dimethyl-alkylammonium chloride and formamide. Rotary platinum shadowing was performed using the Balzers BAF400 with Quartz crystal thin film monitor. Images were acquired on a JOEL 1200 EX transmission electron microscope with side-mounted camera (AMTXR41 supported by AMT software v601) and analyzed with ImageJ (National Institutes of Health).
4 5 8 trimethylpsoralen
Manufactured by Merck Group
4,5′,8-trimethylpsoralen is a chemical compound used in laboratory settings. It is a naturally occurring furanocoumarin derivative. The core function of this compound is to act as a photosensitizer, which can be used in various research and testing applications.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant, by GE Healthcare (2 mentions)
Phosphoimager, by GE Healthcare (2 mentions)
Stratalinker 2400, by Agilent Technologies (2 mentions)
Poly prep chromatography column, by Bio-Rad (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Baf 400, by Oerlikon Balzers (1 mentions)
Genomic tip 20 g column, by Qiagen (1 mentions)
Proteinase k, by Roche (1 mentions)
Deae cellulose, by Merck Group (1 mentions)
5 protocols using 4 5 8 trimethylpsoralen
1
Analyzing DNA Replication Intermediates
2
Stabilizing Replication Intermediates by Psoralen Crosslinking
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hTERT RPE-1 cells were treated as above and harvested. Immediately after, cells were psoralen-crosslinked in vivo to stabilize replication intermediates as described in58 . The cell suspension was first incubated with 30 µg/ml 4, 5′, 8-trimethylpsoralen (2 mg/ml, Sigma) for 5 min in the dark and then exposed to 365 nm UV light for 8 min in a UV Stratalinker 1800, (Stratagene), with 365 nm UV bulbs (model UVL-56, UVP) at 2–3 cm from the light source. The incubation and irradiation steps were repeated three more times (4 cycles total). Genomic DNA (gDNA) was extracted with phenol-chloroform as described in58 . 50 µg of gDNA were digested with KpnI and passed through a QIAGEN Genomic-tip 20/G column (QIAGEN) to enrich for replication intermediates, as described by Zellweger and Lopes59 . EM spreads and imaging was performed as described in60 .
3
Electron Microscopy Analysis of DNA Replication Intermediates
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EM analysis of replication intermediates has been described in detail (Ray Chaudhuri et al., 2012 (link); Neelsen et al., 2014 (link)), including a description of the important parameters to consider specifically for the identification and the scoring of reversed forks (Neelsen et al., 2014 (link)). In brief, 5–10 × 106 U-2 OS cells were harvested and genomic DNA was cross-linked by two rounds of incubation in 10 µg/ml 4,5′,8-trimethylpsoralen (Sigma-Aldrich) and 3 min of irradiation with 366 nm UV light on a precooled metal block. Cells were lysed and genomic DNA was isolated from the nuclei by proteinase K (Roche) digestion and phenol-chloroform extraction. DNA was purified by isopropanol precipitation, digested with PvuII HF in the proper buffer for 3–5 h at 37°C, and replication intermediates were enriched on a benzoylated naphthoylated DEAE–cellulose (Sigma-Aldrich) column. EM samples were prepared by spreading the DNA on carbon-coated grids in the presence of benzyl-dimethyl-alkylammonium chloride and visualized by platinum rotary shadowing. Images were acquired on a transmission electron microscope (JOEL 1200 EX) with side-mounted camera (AMTXR41 supported by AMT software v601) and analyzed with ImageJ (National Institutes of Health).
4
Psoralen Cross-Linking of Genomic DNA
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Psoralen cross-linking12 (link) was performed in isolated nuclei that were irradiated in the presence of 10 μg/ml 4,5,8’-trimethylpsoralen (Sigma–Aldrich). Genomic DNA was isolated and 10 μg was digested with SalI (Promega). Fragmented DNA was separated on a 0.9% agarose gel, transferred onto nylon membranes, and immobilized by UV irradiation at 1875 x 100 μJ/cm2 using a UV cross-linker (Stratalinker 2400; Agilent Technologies). The membrane was hybridized with 32P-labelled rDNA (generated using a nick translation labeling kit, Roche) and visualized on a PhosphoImager (GE Healthcare). Quantification was performed using ImageQuant (TLv2005.04; GE Healthcare). Original psoralen blot scans that correspond to quantitated and/or edited blot images shown in Figs 1 , 2 and Supplementary Fig. 2 are shown in Supplementary Fig. 1a, b and Supplementary Fig. 8 . A representative depiction of the psoralen blot quantitation is shown in Supplementary Fig. 1c, d .
5
Mapping DNA Cross-Linking by Psoralen and UV
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Cells were lysed in 10 mM Tris–HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% NP-40, and nuclei were pelleted, resuspended in 50 mM Tris–HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, and 0.1 mM EDTA, and irradiated in the presence of 4,5,8′-trimethylpsoralen (Sigma-Aldrich) with a 366 nm UV light box at a distance of 6 cm (Conconi et al., 1989 (link)). 200 μg/ml psoralen was added at 1:20 dilution every 4 min for a total irradiation time of 20 min. gDNA was isolated, digested with SalI, and separated on a 0.9% agarose gel, and alkaline Southern blotting was performed. To reverse psoralen cross-linking, filters were treated with 254 nm UV at 1,875 × 100 μJ/cm2 using a UV cross-linker (Stratalinker 2400; Agilent Technologies). The membrane was then hybridized to a purified 32P (Amersham)-labeled rDNA probe (+ 1601-2089), visualized by scanning on a PhosphoImager (GE Healthcare), and quantitated using ImageQuant (TLv2005.04; GE Healthcare).
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