5 ethynyl uridine 5 eu

Manufactured by Jena Biosciences

5-ethynyl uridine (5-EU) is a nucleoside analog that can be used to label and detect newly synthesized RNA in living cells. It is a thymidine analog that can be incorporated into RNA during transcription.

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Lab products found in correlation

Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (1 mentions) Qubit rna hs assay kit, by Thermo Fisher Scientific (1 mentions) Truseq rna sample preparation kit, by Illumina (1 mentions) Tri reagent, by Thermo Fisher Scientific (1 mentions) Copper sulfate, by Merck Group (1 mentions) Atto azide alexa 594, by ATTO-TEC (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Ascorbic acid, by Merck Group (1 mentions) Operetta cls system, by PerkinElmer (1 mentions) Hh17000001, by PerkinElmer (1 mentions) Clk n002 10, by Jena Biosciences (1 mentions)

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5′ethynyl uridine (EU) (6 citations)

4 protocols using 5 ethynyl uridine 5 eu

Nascent RNA Sequencing in Trypanosomes

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Procyclic forms at a density of 7–8 × 10⁶ ml⁻¹ were pulsed for 10 min with 200 μM 5-ethynyl uridine (5-EU; Jena Biosciences). Cells were spun down and nuclei were isolated as described by Murphy et al. (64 (link)) with some modifications. Briefly, cell pellets were resuspended in ice-cold buffer (0.5 M sucrose, 50 mM KCl, 5 mM MgCl₂, 50 mM Tris–HCl, pH 7.4) and incubated on ice. After 5 min, NP-40 was added to final concentration of 0.1%, the sample was vortexed for 10 s and nuclei were pelleted by centrifugation at 3300g at 4°C for 3 min. Total RNA was isolated from nuclear pellets and stored at –70°C. The amount of RNA was measured using a Qubit RNA HS assay kit (Invitrogen). Copper-dependent click-it chemistry was used to label nascent RNAs that incorporated 5-EU with biotin as described (65 (link)), starting with 2 mg total RNA. Biotin-ligated RNA was fragmented to ∼250 bp using a magnesium-dependent RNA fragmentation module (New England Biolabs) and the biotinylated RNA fragments were purified using Dynabeads™ M280 streptavidin (ThermoFisher Scientific) and quantitated using a Qubit RNA HS assay kit. Purified nascent RNAs were used for cDNA library preparation using Illumina Truseq RNA sample preparation kit and sequenced at Fasteris (Geneva) as described above.

Florini F., Naguleswaran A., Gharib W.H., Bringaud F, & Roditi I. (2018). Unexpected diversity in eukaryotic transcription revealed by the retrotransposon hotspot family of Trypanosoma brucei. Nucleic Acids Research, 47(4), 1725-1739.

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Measuring RNA Synthesis Dynamics

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Cells from each line were plated onto 500 cm² plates at 6.6 million cells per plate and cultured for two days such that they reached ~70%–80% confluency, at which point growth media was supplemented with 5-ethynyl uridine (5EU, Jena Biosciences) (Jao and Salic, 2008 (link)) at a final concentration of 400 μM. After the desired labeling intervals cells were harvested (Figure 1A). Four plates were harvested for each 40 min time interval, three plates for each 1 h time interval, and two plates for each other time interval. A plate that had never received 5EU was harvested in parallel for each condition.
Cells were harvested at 4°C, washed twice with 50 mL ice-cold PBS, pH 7.3 containing 100 μg/mL cycloheximide and then used to prepare cytoplasmically enriched lysate as described (Subtelny et al., 2014 (link)). An aliquot of cleared lysate was flash frozen for use in ribosome profiling (Eisen et al., 2020 ), and the rest of the lysate was added to 5 volumes of TRI reagent (Ambion) and frozen at −80°C. Samples stored in TRI reagent were thawed at room temperature, and RNA was purified according to the manufacturer’s protocol and used for RNA-seq or PAL-seq v2.

Eisen T.J., Eichhorn S.W., Subtelny A.O., Lin K.S., McGeary S.E., Gupta S, & Bartel D.P. (2020). The Dynamics of Cytoplasmic mRNA Metabolism. Molecular cell, 77(4), 786-799.e10.

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Visualizing Nascent RNA Dynamics

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Cells were irradiated with UV-C light (9 J m⁻² or 12 J m⁻²), allowed to recover for the indicated periods and pulse labeled with 400 µM 5-ethynyl-uridine (5-EU; Jena Bioscience) for 1 h, followed by a 15-min medium chase with DMEM without supplements. Cells were fixed with 3.7% formaldehyde in PBS for 15 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature and blocked in 1.5% BSA (Thermo Fisher Scientific) in PBS. Nascent RNA was visualized by click-it chemistry, labeling the cells for 1 h with a mix of 60 µM Atto azide-Alexa 594 (Atto Tec), 4 mM copper sulfate (Sigma-Aldrich), 10 mM ascorbic acid (Sigma-Aldrich) and 0.1 μg ml⁻¹ DAPI in a 50 mM Tris buffer (pH 8). Cells were washed extensively with PBS and mounted in Polymount (Brunschwig).

Kokic G., Yakoub G., van den Heuvel D., Wondergem A.P., van der Meer P.J., van der Weegen Y., Chernev A., Fianu I., Fokkens T.J., Lorenz S., Urlaub H., Cramer P, & Luijsterburg M.S. (2024). Structural basis for RNA polymerase II ubiquitylation and inactivation in transcription-coupled repair. Nature Structural & Molecular Biology, 31(3), 536-547.

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Quantifying RNA Synthesis in Cells

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Cells were seeded at a cell density of 5 × 10⁴ cells mL⁻¹. The next day cells were treated with Osm^G and Osm^M. To measure RNA synthesis, 1 µm of 5‐Ethynyl‐uridine (5‐EU, Jena Bioscience, and CLK‐N002‐10) was added to each condition for 2 h. Next, cells were washed and fixed with PFA 4% for 15 min. Subsequently, cells were permeabilized with Triton X‐100 0.5% for 20 min at room temperature and incubated with the Click‐it mix (PBS, CuSO₄ (100 µm), Azide (200 µm) and Sodium Ascorbate (1 m)) for 15 min. After washing, cells were incubated with DAPI for 10 min. Imaging was performed using the High Throughput Imager Operetta CLS system (Perkin Elmer, HH16000000), while image analysis and quantification were performed with the Harmony 4.2 software (Perkin Elmer, HH17000001).

Chui J.S., Izuel‐Idoype T., Qualizza A., de Almeida R.P., Piessens L., van der Veer B.K., Vanmarcke G., Malesa A., Athanasouli P., Boon R., Vriens J., van Grunsven L., Koh K.P., Verfaillie C.M, & Lluis F. (2023). Osmolar Modulation Drives Reversible Cell Cycle Exit and Human Pluripotent Cell Differentiation via NF‐κВ and WNT Signaling. Advanced Science, 11(7), 2307554.

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