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Trimethylpsoralen

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Trimethylpsoralen is a psoralen compound used in various laboratory applications. It is a photosensitizing agent that can interact with nucleic acids upon exposure to ultraviolet light. The core function of Trimethylpsoralen is to facilitate the study of DNA and RNA-related processes in controlled experimental settings.

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Lab products found in correlation

Ace600 coating system, by Leica (2 mentions) 2k ccd camera, by Advanced Microscopy Techniques (2 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions)

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4,5′,8-trimethylpsoralen (5 citations)

4 protocols using trimethylpsoralen

1

Generating C. elegans Strains Expressing TDP-1

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All C. elegans strains are on the N2 Bristol background and cultured under standard conditions at 20°C unless otherwise indicated. To generate the Psnb-1::TDP-1-YFP(iwIs53) strain, a transgene DNA construct was generated by subcloning TDP-1 cDNA with a C-terminal YFP tag into a modified plasmid, pPD30_38 (Fire Lab Vector, Addgene), with the promoter replaced with that of the snb-1 gene, as previous described [60] (link). The transgene DNA solution containing 20 ng/µl of the expression construct was injected into hermaphrodite gonads [79] (link), and multiple extrachromosomal lines were established based upon the fluorescent markers. These lines were further treated with 30 µg/ml trimethylpsoralen (Sigma-aldrich) and 300 µJ of 365 nm UV light to screen for integrated lines that stably expressed the transgenes. Each integrated line was backcrossed with the N2 strain at least four times. The Psnb-1::TDP-C25-YFP(iwIs22) strain was reported previously [60] (link). Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).
Zhang T., Baldie G., Periz G, & Wang J. (2014). RNA-Processing Protein TDP-43 Regulates FOXO-Dependent Protein Quality Control in Stress Response. PLoS Genetics, 10(10), e1004693.
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2

Aphidicolin-Mediated Cell Cycle Arrest

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One day after addition of N3 media (and without addition of glial cultures), cells were treated with 1 μM aphidicolin for 1.5–2 hours in N3 media. Cells were then trypsinized in 0.25% Trypsin-EDTA, spun down, and resuspended in complete N3 media + 1 μM aphidicolin + 2 μg/mL trimethylpsoralen (Sigma). 500 μL of control-puro was removed and saved for the no UV crosslinking control. Each 1mL of the remaining samples were added to individual wells of a 24-well plate and then exposed to 3kJ m-2 of 365nM light (Fotodyne UV Transilluminator 3–3000 with 15W bulbs) for 15 minutes at room temperature in the dark. Cells were then re-collected, spun at 1000 × g for 5 minutes, washed with 1mL PBS, and spun down again. Then cells were resuspended in 200 μL PBS and purified using QIAGEN DNeasy Blood and Tissue Kit with inclusion of an RNase A digestion (QIAGEN Cat. No: 69504). Samples were eluted once in 200 μL followed by a second elution in 100 μL of Buffer AE and eluates were then combined.
Babos K.N., Galloway K.E., Kisler K., Zitting M., Li Y., Shi Y., Quintino B., Chow R.H., Zlokovic B.V, & Ichida J.K. (2019). Mitigating Antagonism between Transcription and Proliferation Allows Near-Deterministic Cellular Reprogramming. Cell stem cell, 25(4), 486-500.e9.
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3

Electron Microscopy of Replication Intermediates

Cited 1 time
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Electron microscopy analysis of the replication intermediates was performed as previously described7 (link). Briefly, replication reactions were stopped at 90 min with 10 volumes of Stop Solution C (100 mM Tris-HCl [pH 7.5], 6.7 mM MgCl2, 1 mM EDTA [pH 8.0], and 1% SDS). The DNA was crosslinked with trimethylpsoralen (Sigma) and irradiation with UV light at 365 nM prior to protein extraction and DNA purification. Purified DNA was incubated with E. coli single-stranded DNA binding protein (SSB), fixed with 0.3% glutaraldehyde, then purified by size-exclusion chromatography. Eluted complexes were mounted onto grids, which were then subjected to rotary shadowing with platinum and carbon coating using a Leica Ace600 coating system. Samples were imaged using a JEOL 1200EX transmission electron microscope equipped with a 2k CCD camera (Advanced Microscopy Techniques). After blinding the scorer to the conditions, reversed forks were counted and expressed as a percentage of pre-incision structures, which was then normalized to the mock-depleted condition.
Wu R.A., Semlow D.R., Kamimae-Lanning A.N., Kochenova O.V., Chistol G., Hodskinson M.R., Amunugama R., Sparks J.L., Wang M., Deng L., Mimoso C.A., Low E., Patel K.J, & Walter J.C. (2019). TRAIP is a master regulator of DNA interstrand cross-link repair. Nature, 567(7747), 267-272.
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4

Electron Microscopy of Replication Intermediates

Cited 1 time
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Electron microscopy analysis of the replication intermediates was performed as previously described7 (link). Briefly, replication reactions were stopped at 90 min with 10 volumes of Stop Solution C (100 mM Tris-HCl [pH 7.5], 6.7 mM MgCl2, 1 mM EDTA [pH 8.0], and 1% SDS). The DNA was crosslinked with trimethylpsoralen (Sigma) and irradiation with UV light at 365 nM prior to protein extraction and DNA purification. Purified DNA was incubated with E. coli single-stranded DNA binding protein (SSB), fixed with 0.3% glutaraldehyde, then purified by size-exclusion chromatography. Eluted complexes were mounted onto grids, which were then subjected to rotary shadowing with platinum and carbon coating using a Leica Ace600 coating system. Samples were imaged using a JEOL 1200EX transmission electron microscope equipped with a 2k CCD camera (Advanced Microscopy Techniques). After blinding the scorer to the conditions, reversed forks were counted and expressed as a percentage of pre-incision structures, which was then normalized to the mock-depleted condition.
Wu R.A., Semlow D.R., Kamimae-Lanning A.N., Kochenova O.V., Chistol G., Hodskinson M.R., Amunugama R., Sparks J.L., Wang M., Deng L., Mimoso C.A., Low E., Patel K.J, & Walter J.C. (2019). TRAIP is a master regulator of DNA interstrand cross-link repair. Nature, 567(7747), 267-272.
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