For the RRS following DRB treatment, cells were treated with 100 μM DRB (Sigma, D1916) for 2 h and either during the DRB treatment or following DRB washout the cells were pulse-labelled with 400 μM 5-ethynyl-uridine (EU; Jena Bioscience) for 1 h. For the RRS following UV irradiation, cells were irradiated with UV-C light (9 J/m2 or 12 J/m2), allowed to recover for the indicated time periods, and pulse-labelled with 400 μM 5-ethynyl-uridine (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 RT and blocked in 1.5% bovine serum albumin (BSA, Thermo Fisher) in PBS. Nascent RNA was visualized by click-it chemistry, labelling the cells for 1 h with a mix of 60 μM Atto azide-Alexa594 (Atto Tec), 4 mM copper sulfate (Sigma), 10 mM ascorbic acid (Sigma) and 0.1 μg/mL DAPI in a 50 mM Tris-buffer. Cells were washed extensively with PBS and mounted in Polymount (Brunschwig).
5 ethynyl uridine
Manufactured by Jena Biosciences
5'-Ethynyl uridine (EU) is a nucleoside analog that can be used to label newly synthesized RNA in living cells. It is a structural analog of uridine, with an ethynyl group attached to the 5' position of the uridine base. This modification allows for the detection and visualization of RNA synthesis by click chemistry reactions.
Automatically generated - may contain errors
Lab products found in correlation
Ascorbic acid, by Merck Group (2 mentions)
Cuso4, by Merck Group (2 mentions)
Copper sulfate, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Atto azide alexa 594, by ATTO-TEC (1 mentions)
Qiashredder, by Qiagen (1 mentions)
Turbo dnase treatment, by Thermo Fisher Scientific (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Dialyzed fetal calf serum, by Thermo Fisher Scientific (1 mentions)
Ham s f10 medium, by Thermo Fisher Scientific (1 mentions)
Atto 594 azide, by ATTO-TEC (1 mentions)
Lsm 700 axio imager z2, by Zeiss (1 mentions)
Formaldehyde fa, by Merck Group (1 mentions)
Alexa fluor 488 conjugated azide, by Thermo Fisher Scientific (1 mentions)
Tris base, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
6 protocols using 5 ethynyl uridine
1
Visualizing Nascent RNA Kinetics
2
Transcriptome Labeling and Sequencing
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SARS-Cov2 or mock infected Calu3 cells and Nsp16- or GAPDH-expressing HEK293Ts were labeled with 5-Ethynyl-uridine (5EU; Jena Bioscience) by adding 5EU containing media to cells for 20 min at a final concentration of 1mM, as previously described (Jao and Salic, 2008 (link)). After the pulse label, cells were washed with warm PBS and lysed in RLT buffer (QIAGEN). Total RNA was isolated from cells using manufacturer’s protocols for Qiashredder and RNeasy RNA isolation (both QIAGEN), followed by Turbo DNase treatment (Ambion, Thermo Scientific), and Zymo RNA Clean and Concentrate. For each sample, 2 μg of RNA was used for ligation of a unique barcoded RNA adaptor, following the relevant steps in the protocol described above in Library Construction of RNA-seq libraries. Samples were then pooled before proceeding to biotinylation steps.
3
Measuring Transcription Levels by EU Labeling
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Transcription levels were measured by pulse labeling with the nucleotide analogue 5′ethynyl uridine (EU) (Jena Bioscience). Cells were grown to 80% confluency on glass coverslips, treated as indicated, and incubated for 30 min with 1 µM EU in Ham’s F10 medium supplemented with 10% dialyzed fetal calf serum (Gibco). Subsequently, cells were washed with PBS, fixed with 2% PFA in PBS for 15 min. After permeabilisation with 0.1% triton in PBS for 10 min, click chemistry-based azide coupling was performed by incubation for 30 min with 60 µM Atto594 Azide (Attotec, Germany) in 50 mM Tris buffer (pH 8) with 4 mM CuSO4 (Sigma Aldrich) and 10 mM freshly prepared ascorbic acid. Coverslips were washed with PBS and mounted with Vectashield containing DAPI (Brunschwig Chemie). All steps were performed at RT. Cells were imaged with a Zeiss LSM 700 Axio Imager Z2 upright microscope equipped with a 63x Plan-Apochromat oil immersion lens (NA 1.40) using Carl Zeiss LSM (version 14.0.0.0) and image analysis was performed using Image J.
4
Labeling Nascent DNA and RNA in Mice
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Replicating cells and nascent mtDNA were labeled by administering a dose of 100 mg/kg body weight BrdU (Sigma-Aldrich, #B5002) via intraperitoneal injection to P28-29 mice. Tissue samples were collected 16 h later. The samples from BrdU-exposed mice were not used for DNA damage assays.
Nascent RNA was labeled by intraperitoneal injection of 88 mg/kg 5-ethynyl-uridine (EU) (Jena Biosciences, #CLK-N002-10) to P25 mice. Tissue samples were collected 6 h later.
Nascent RNA was labeled by intraperitoneal injection of 88 mg/kg 5-ethynyl-uridine (EU) (Jena Biosciences, #CLK-N002-10) to P25 mice. Tissue samples were collected 6 h later.
5
Measuring Transcription Recovery After UV
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Cells were seeded on coverslips at least 2 days prior to experiments. Seeded cells were pulse-labeled with 200 mM 5′ethynyl uridine (EU, Jena Bioscience) in culture medium for 30 min before fixation with 3.7% formaldehyde (FA, Sigma) at room temperature for 15 min. After permeabilization with 0.5% Triton-100 in PBS, Click-iT azide-based reaction was performed as described in the manufacturer’s manual. DAPI was added to visualize the nuclei. Images were captured using a Zeiss LSM 700 confocal and quantified by ImageJ as integrated intensity.
In order to measure the recovery of transcription rate after UV, cells were mock-treated or irradiated with 8J/m2 UV-C, 2 or 24 h before EU incorporation.
In order to measure the recovery of transcription rate after UV, cells were mock-treated or irradiated with 8J/m2 UV-C, 2 or 24 h before EU incorporation.
6
mRNA Labeling and Immunofluorescence Staining
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To label nascent mRNA, cells were pulsed and labeled as previously described (Jao and Salic, 2008 (link)), and following manufacturer’s protocols. Cells migrating in microfluidic devices were pulsed with 1 mM 5-ethynyl uridine (EU, Jena Bioscience) in complete DMEM for 4 h, and then fixed with 4% PFA in PBS for 30 min. After two PBS washes, the devices were carefully removed as described in the IF staining section. The cells were then permeabilized with 0.3% Triton X-100 (Sigma) in PBS for 15 min and incubated for 30 min with freshly prepared EU labeling buffer containing 100 mM Tris base (Sigma), 100 μM Alexa Fluor 488 conjugated-azide (Invitrogen), 4 mM CuSO4 (Sigma), and 100 mM ascorbic acid (Sigma) in water. After the incubation, cells were washed with PBS, and another round of 30 min incubation with EU labeling buffer and PBS washes was repeated. Cells were then incubated with primary antibodies and proceeded with the IF staining protocol.
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