Name: Metabolic Labeling of the Nascent Transcriptome in Xenopus Early Embryogenesis (Video) | JoVE Journal
Uploaded: 2026-03-27T14:00:00.000+00:00
Duration: 6 min 46 s
Description: Learn metabolic labeling of nascent transcripts during zygotic genome activation in Xenopus laevis embryos using 5-EU microinjection. This sensitive method enables precise temporal profiling of early gene activation dynamics.

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5 minutes

We thank the Matthew Good lab at the University of Pennsylvania for providing training. This work was supported in part by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R03HD105802).

Animal work described in this study has been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of South Carolina. Laboratory safety and waste disposal should adhere to institutional regulations.
1. Preparation of Reagents and Buffers
<ol>
	<li>20x MMR (Marc&#39;s Modified Ringer) solution: Mix 100 mM HEPES, 2 mM EDTA, 2 M NaCl, 40 mM KCl, 20 mM MgCl2, and 40 mM CaCl2. Adjust pH to 7.8 with NaOH. Autoclave and store at room temperature.</li>
	<li>3% Ficoll (w/v)/0.5x MMR: Slowly add 1.5 g of Ficoll 400 into 50 mL of 0.5x MMR while stirring gently with a magnetic stirring bar until the Ficoll is completely dissolved. Autoclave and keep it at 4 &#176;C until use.</li>
	<li>5-ethynyl uridine (5-EU) stock solution: To make the 50 mM stock solution, add 372.8 &#181;L of buffer (note that any buffer compatible with microinjection can be used, e.g., 1x TBS, pH 7.6) into the 5-mg vial. Mix well and make 2 &#181;L aliquots. Store at -80 &#176;C.</li>
</ol>
2. Microinjection of 5-EU into 1-cell embryos
<ol>
	<li>To obtain Xenopus embryos, follow standard protocols to perform in vitro fertilization (IVF) (e.g., see Sive et al., 2007)16. The embryo should be in 0.1x MMR, ready for use.</li>
	<li>Add 1 mL of 3% Ficoll/0.5x MMR into a microinjection chamber made from a standard glass slide (25 mm x 75 mm). Use a plastic transfer pipette to transfer 20 (or more if needed) embryos into the center of the microinjection chamber.</li>
	<li>Use a fine tweezer to carefully cut the very thin tip end of the glass needle and stick the needle vertically (with its tip end facing down) against the modeling clay on the wall of the bench. Use the P2 pipette with the microloader tip to add 0.5 &#181;L of 50 mM 5-EU from the top of the glass needle.</li>
	<li>Assemble the needle onto a micromanipulator. Use the P10 pipette to add 10 &#181;L of halocarbon oil on a stage micrometer and put it under a stereomicroscope.</li>
	<li>To calibrate the injection volume, first use the foot to press the pad on the floor linked to the microinjector and inject a droplet into the halocarbon oil on the micrometer. Depending on the droplet size, adjust the pressure and injection time on the microinjector to determine the injection volume to be 10 nL, i.e., 0.27 mm in diameter on the micrometer.</li>
	<li>Use a tweezer in one hand to help hold the embryo, use the other hand to hold the micromanipulator, and inject 10 nL of 50 mM EU into embryos at the 1-cell stage. The final concentration of 5-EU in the embryo is ~0.5 mM.</li>
	<li>After microinjection, use a plastic transfer pipette to transfer the embryos from the injection chamber into a new glass dish containing 3% Ficoll/0.5x MMR. Keep the injected embryos in Ficoll for ~1 h.</li>
	<li>Carefully remove the Ficoll solution and replace the medium with 0.1x MMR. Keep embryos in 0.1x MMR until further use. Constantly monitor the development of the embryos and remove any abnormal embryos if necessary.</li>
	<li>When the embryos reach the desired stages of development, use a plastic transfer pipette to transfer 10 embryos into a 1.5 mL microcentrifuge tube. Use a P200 pipette to carefully remove any residual medium in the tube, and snap freeze the embryos in liquid nitrogen. 
	Note: To ensure the rigor and reproducibility, only collect the embryos that are normal based on their appearance as compared with the control. Remove and exclude any abnormal embryos from further analysis. If needed, also collect the same number of embryos without microinjection as negative controls.</li>
	<li>Store the embryos in a -80 &#176;C freezer until further use.</li>
</ol>
3. Total RNA Extraction and Concentration Measurement
NOTE: Use a silica membrane&#8211;based total RNA extraction kit to isolate total RNAs from early embryos and a fluorescence-based high-sensitivity RNA quantification kit to determine the concentrations of RNAs. Follow the instructions provided by the kit, and the following steps are slightly modified from the manual. All solutions and reagents used in the following steps should be RNase-free.
<ol>
	<li>Put the samples from the -80 &#176;C freezer on ice.</li>
	<li>Add 700 &#181;L of the Buffer RLT from the RNA extraction kit in each tube. Use a P1000 pipette to pipette up and down for at least 15 times or until all embryos are dissolved.</li>
	<li>Keep the samples on ice for 20 min. Gently mix again by pipetting up and down for at least 10 times to completely homogenize the embryos.</li>
	<li>Add 700 &#181;L of 70% ethanol to the homogenized embryos. Pipette up and down to mix well and transfer the 700 &#181;L of the mixture into a spin column.</li>
	<li>Centrifuge the column at 10,000 x g for 1 min. Discard the flow-through. Repeat the centrifuge until the rest of the mixture is complete.</li>
	<li>Add 350 &#181;L of the Buffer RW1 and centrifuge the column at 10,000 x g for 1 min. Discard the flowthrough.</li>
	<li>Carefully add 80 &#181;L of DNase I to the center of the membrane in the column. Incubate for 15 min at room temperature.</li>
	<li>Add 350 &#181;L of Buffer RW1 and centrifuge the column at 10,000 x g for 1 min. Discard the flowthrough.</li>
	<li>Add 500 &#181;L of Buffer RPE and centrifuge the column at 10,000 x g for 1 min. Discard the flowthrough. Repeat the washing once more.</li>
	<li>Dry the column by centrifuging it again at 10,000 x g for 2 min.</li>
	<li>Transfer the column to a new RNase-free tube. Carefully add 35 &#181;L of RNase-free H2O to the center of the membrane in the column.</li>
	<li>Centrifuge the column at 10,000 x g for 1 min. Collect the eluted RNA and keep on ice.</li>
	<li>To measure the RNA concentration, first prepare the working solution using the RNA quantification kit by diluting the RNA reagent (dye) at 1:200 in the RNA buffer. Calculate the total volume of the working solution depending on the total number of standards and prepare 200 &#181;L for each standard or sample.</li>
	<li>Set up a 0.2-mL 8-tube strip. To dilute each standard, add 10 &#181;L to 190 &#181;L of the working solution. To dilute each sample, add 1 &#181;L to 199 &#181;L of the working solution. Mix well.</li>
	<li>Put the 8-tube strip in a fluorometer. Select the RNA Assay, calibrate with the standards, and read the concentrations of the samples.</li>
</ol>
4. Nascent EU-RNA Biotinylation and Purification
NOTE: A click chemistry&#8211;based nascent RNA capture kit is used to biotinylate and pull down the nascent EU-RNAs. Follow the instructions provided by the kit, and the following steps are slightly modified. RNAs from non-injected embryos, as well as EU-RNAs without biotinylation, can be served as background controls for pulldown specificity.
<ol>
	<li>RNA Biotinylation and Cleaning
	<ol>
		<li>For each sample, mix the following components in a 1.5 mL microcentrifuge tube to make a 50-&#181;L click reaction: total RNA (1&#8211;10 &#181;g), 1x EU buffer, 2 mM CuSO4, 0.25 mM biotin azide, 10 mM additive 1, and 12 mM additive 2.</li>
		<li>Incubate the reaction mix at room temperature for 30 min on a nutator.</li>
		<li>In each reaction above, add 1 &#181;L of glycogen, 50 &#181;L of 7.5 M ammonium acetate, and 700 &#181;L of chilled 100% ethanol. Pipette up and down 5 times to mix them well.</li>
		<li>Put the tube in a -80 &#176;C freezer and keep it overnight.</li>
		<li>Centrifuge the tube at 13,000 x g for 20 min at 4 &#176;C. Carefully remove the supernatant without disturbing the pellet.</li>
		<li>Add 700 &#181;L of 75% ethanol and mix gently to resuspend the pellet. Centrifuge the tube at 13,000 x g for 5 min at 4 &#176;C. Carefully remove the supernatant without disturbing the pellet. Repeat the washing once more.</li>
		<li>Air dry the pellet for 5&#8211;10 min at room temperature. Resuspend the pellet in 10 &#181;L RNase-free H2O.</li>
	</ol>
	</li>
	<li>EU-RNA Purification
	<ol>
		<li>To pull down the nascent EU-RNAs, for each sample, dispense 5 &#181;L of streptavidin-coated magnetic beads in a new 1.5 mL microcentrifuge tube.</li>
		<li>Add 9 &#181;L of Wash Buffer 2. Mix well and place the tube on a magnetic rack. Let it stand for 2 min. Remove the supernatant. Repeat the washing for an additional two times. Resuspend the beads in 5 &#181;L of Wash Buffer 2.</li>
		<li>Prepare the following master mix in a new 1.5 mL microcentrifuge tube (15 &#181;L for each sample): 12.5 &#181;L of RNA binding buffer (Component G), 0.2 &#181;L of 1:10 diluted RNase OUT, and 2.3 &#181;L of RNase-free H2O. Add 15 &#181;L of the mix to each tube containing the RNA sample.</li>
		<li>Incubate for 5 min on a heat block at 69 &#176;C. Put the tube on ice.</li>
		<li>Add 5 &#181;L of the above pre-washed beads to each tube containing the RNA sample. Incubate for 30 min on a nutator at room temperature.</li>
		<li>Add 50 &#181;L of Wash Buffer 1. Mix well and place the tube on a magnetic rack. Let it stand for 2 min. Remove the supernatant. Repeat the washing another 4 times. 
		Note: The supernatant after the first wash, which presumably contains only the maternal transcripts, can be collected for measuring the maternal transcriptome.</li>
		<li>Add 50 &#181;L of Wash Buffer 2. Mix well and place the tube on a magnetic rack. Let it stand for 2 min. Remove the supernatant. Repeat the washing another 4 times.</li>
		<li>Resuspend the beads in 5 &#181;L of Wash Buffer 2. The beads, which contain the nascent EU-RNAs, are ready for direct cDNA synthesis and library prep. 
		Note: Before proceeding to library preparation, the enrichment of nascent transcripts can be assessed by measuring expression of known individual genes using reverse transcription-polymerase chain reaction (RT-PCR).</li>
	</ol>
	</li>
</ol>
5. RNA Library Preparation and Sequencing
<ol>
	<li>Use commercially available kits to prepare the cDNA libraries and follow the instructions provided by the kit. Use the NEBNext Library Quant Kit for Illumina to quantify the libraries and pool them.</li>
	<li>Pool the libraries following the instructions of the commonly used next-generation sequencing kits. Dilute the libraries to ~1.8 pM before loading them into the benchtop sequencer to achieve an optimal cluster density of ~200k/mm2 during sequencing. Sequence the libraries using the commonly used next-generation sequencing. Used paired-end 75-bp sequencing for ~20 million reads per sample.</li>
	<li>Quantify the transcriptomic reads by aligning the sequences to the Xenopus laevis genome build 9.2 using salmon17 and perform differential gene expression analysis using DESeq218. To visualize the peaks in the genome browser, map the sequences to the genome using STAR19 and display in the Integrative Genomics Viewer (IGV)19.</li>
</ol>

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<li>Chen, H., Einstein, L. C., Little, S. C., Good, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatiotemporal+patterning+of+zygotic+genome+activation+in+a+model+vertebrate+embryo%5BTitle%5D)+AND+%22Dev+Cell%22%5BJournal%5D)">Spatiotemporal patterning of zygotic genome activation in a model vertebrate embryo.</a> Dev Cell. 49 (6), 852-866.e7 (2019).</li>
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<li>Palozola, K. C., Donahue, G., Zaret, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Eu-rna-seq+for+in+vivo+labeling+and+high+throughput+sequencing+of+nascent+transcripts%5BTitle%5D)+AND+%22STAR+Protoc%22%5BJournal%5D)">Eu-rna-seq for in vivo labeling and high throughput sequencing of nascent transcripts.</a> STAR Protoc. 2 (3), 100651(2021).</li>
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The author declares no competing interests.

Here we have provided a detailed protocol for metabolic labeling the nascent transcriptome in early embryogenesis, including microinjecting the 5-EU into 1-cell embryos, extracting total RNAs and measuring their concentrations, and biotinylating and purifying the nascent EU-RNAs for library preparation and sequencing (EU-RNA-Seq) (Figure 1). Because of the high sensitivity and specificity of the approach compared with total RNA-seq, this protocol allowed us not only to accurately determine b...

Following fertilization, the embryo is under the control of maternal factors deposited in the egg, while the zygotic genome is transcriptionally silent for a period; it is activated to transcribe hundreds to thousands of genes that are essential for subsequent development, including cell fate specification and germ layer formation1,2,3,4,5,6. This phenomenon of zygotic genome activation (ZGA) during the maternal-to-zygotic transition (MZT) is highly conserved in all metazoan species, although the timing of ZGA varies among species1,2,3,4,5,6. Understanding how ZGA is regulated is not only important for our understanding of the fundamental principles of early development but also offers potential for developing novel strategies for diagnosing and treating developmental disorders and increasing reproductive health.
A major challenge of studying ZGA has been to accurately detect new transcripts and determine their onset time during zygotic genome activation (ZGA). Due to the predominance of maternal transcripts in early embryos7, the high levels of these transcripts can mask the detection of new transcripts during ZGA, particularly for those lowly activated genes, using conventional bulk RNA-seq. To overcome this challenge, we developed a method to measure the nascent transcriptome by using metabolic labeling of the nascent transcripts with 5-ethynyl-uridine (5-EU), followed by biotinylation of the nascent EU-RNAs via click chemistry and purifying them for sequencing (EU-RNA-seq)8,9,10. As compared with other nascent transcript profiling methods, such as those using 4-thiouridine (4sU)11,12, 5-EU labeling is highly specific via click chemistry, can be used for both imaging and sequencing, and does not require chemical conversion. Moreover, the nascent EU-RNA-seq is highly sensitive and detects hundreds of genes, the activation of which cannot be detected by the conventional bulk RNA-seq8. This approach also allowed us to accurately determine the onset of individual zygotic gene activation and profile the distinct spatiotemporal patterns of ZGA during early embryogenesis8.
We describe here the detailed procedures for preparing samples for the EU-RNA-seq to profile the nascent transcriptome using Xenopus laevis early embryos as a model. This protocol is adapted from a previous method10,13 with minor optimizations in the procedures, including the microinjection of 5-EU into the 1-cell stage embryos, biotinylation of nascent EU-RNAs via click reaction, and purifying them for sequencing (Figure 1). To ensure reproducibility, depending on the stages of early embryos with varying abundance of nascent transcripts, 1&#8211;10 &#181;g of total RNA should be used as input, although a lower amount can still be used. This protocol can be applied in other embryonic systems, such as Drosophila14 and zebrafish15, and other tissues13. This method is a powerful tool for our understanding of gene transcription and regulatory mechanisms in various biological processes, including revealing novel principles of early embryogenesis.

<table><tbody><tr><td>&amp;micro;PUPM Microinjector</td><td>World Precision Instruments</td><td>UPUMP</td><td>For microinjection (microinjecting with pressure)</td></tr><tr><td>5-ehtynyl uridine</td><td>Thermo Fisher Scientific</td><td>E10345</td><td>For microinjection (labeling nascent traqnscripts)</td></tr><tr><td>ACCU-SCOPE 3075 Zoom Stereomicroscopes</td><td>VWR</td><td>470351-130</td><td>For microinjection (visualizing embryos)</td></tr><tr><td>Ammonium acetate</td><td>Sigma-Aldrich</td><td>A2706</td><td>For RNA precipitation</td></tr><tr><td>Ascorbic acid</td><td>Sigma-Aldrich</td><td>A7506</td><td>For click reaction</td></tr><tr><td>Borosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>TW100-4</td><td>For microinjection (making glass needles)</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5425R</td><td>For centrifuge</td></tr><tr><td>Click-iT Nascent RNA Capture Kit</td><td>Thermo Fisher Scientific</td><td>C10365</td><td>For biotinylating and purifying nascent RNA</td></tr><tr><td>CuSO4</td><td>Sigma-Aldrich</td><td>61230</td><td>For click reaction</td></tr><tr><td>DNase I</td><td>Roche Diagnostics</td><td>4716728001</td><td>For RNA extraction</td></tr><tr><td>Ficoll 400</td><td>Sigma-Aldrich</td><td>GE17-0300-10</td><td>For microinjection (incubating embryos)</td></tr><tr><td>Glasss Petri Dish (60-mm)</td><td>VWR</td><td>75845-542</td><td>For fertilization</td></tr><tr><td>Glycogen</td><td>Thermo Fisher Scientific</td><td>AM9510</td><td>For RNA precipitation</td></tr><tr><td>Halocarbon Oil 27</td><td>Sigma-Aldrich</td><td>H8773</td><td>For microinjection (calibrating injection volume)</td></tr><tr><td>L-cysteine</td><td>Sigma-Aldrich</td><td>168149</td><td>For dejellying embryos</td></tr><tr><td>Microloader tips</td><td>calibre scientific</td><td>EPE-5242956003</td><td>For microinjection (loading 5-EU into the glass needle)</td></tr><tr><td>NEBNext Library Quant Kit for Illumina</td><td>NEB</td><td>E7630</td><td>For library quantification</td></tr><tr><td>NSQ 500/550 Hi Output KT v2.5 (75 CYS)</td><td>Illumina</td><td>20024906</td><td>For sequencing</td></tr><tr><td>Qubi Flex Fluorometer</td><td>Thermo Fisher Scientific</td><td>Q33327</td><td>For measuring RNA concentration</td></tr><tr><td>Qubit RNA HS Binding Kit</td><td>Thermo Fisher Scientific</td><td>Q32852</td><td>For measuring RNA concentration</td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td>For RNA extraction</td></tr><tr><td>Stage Micrometer</td><td>Ward&#039;s Science</td><td>470175-914</td><td>For microinjection (calibrating injection volume)</td></tr><tr><td>Universal RNA-seq with NuQuant</td><td>NuGEN</td><td>0364</td><td>For library preparation</td></tr></tbody></table>

metabolic labeling of the nascent transcriptome in xenopus early embryogenesis

Metabolic Labeling of the Nascent Transcriptome in Xenopus Early Embryogenesis

Early embryogenesis requires new transcription of zygotic genes from the initially dormant zygotic genome after fertilization. A major challenge has been to accurately detect the new transcripts and determine their time of expression during zygotic genome activation (ZGA). Here, we provide detailed procedures for metabolic labeling of the nascent transcripts by 5-ethynyl-uridine (5-EU) in early Xenopus laevis embryos. After fertilization, 5-EU is microinjected into the 1-cell stage embryos to metabolically label the newly transcribed RNAs during ZGA. The nascent EU-RNAs are subsequently purified after biotinylation via click chemistry-mediated reactions, and the nascent transcriptome is determined using next-generation sequencing. Importantly, this method has high sensitivity and specificity that allow the characterization of lowly activated genes. Thus, this approach provides a powerful tool to study the dynamics of gene transcription during early development, and it can be generalized to other embryonic systems or tissues.

Xenopus laevis embryos (four biological replicates) at the stages from 5 to 9 hours post-fertilization (hpf) at 22 °C, which covers the stages from Stage 5 (with little large-scale ZGA) to Stage 9 (with high ZGA), were processed following the protocol above8. The total RNAs, the purified nascent EU-RNAs, and the flow-through RNAs were used for preparing the libraries and were sequenced. As shown in Figure 2, by comparing the nascent transcriptome at 6–9 hpf w...

We provide detailed methods to metabolically label and purify nascent transcripts for transcriptome analysis in Xenopus early embryos using 5-ethynyl-uridine (5-EU).

Early embryogenesis requires new transcription of zygotic genes from the initially dormant zygotic genome after fertilization. A major challenge has been ...

In this study, we investigate how zygotic gene activation and the transitional regulation occur during early xenopus embryo genesis. Existing methods struggle to detect the newly activated zygotic transcripts emit abundant maternal RNAs. This protocol labels and enriches nascent transcripts.To begin, place the microinjection chamber under a stereo microscope and add one milliliter of Ficoll solution into the chamber. Obtain Xenopus laevis embryos stored in 0.1X marks modified ringers or MMR solution. Use a plastic transfer pipette to transfer 20 or more embryos to the center of the microinjection chamber.Prepare the injection needle by cutting the fine tip of a glass needle with tweezers. Position the needle vertically with the tip facing down and load 0.5 microliters of 50 mM 5-ethynyluridine or 5 M EU using a P2 pipette with a micro loader tip. Mount the needle onto a micro manipulator.Using a P10 pipette, place 10 microliters of halocarbon oil on a stage micrometer positioned under a stereo microscope. For volume calibration, press the micro injector foot pedal and inject a droplet into the halocarbon oil on the micrometer. Adjust the pressure and injection time on the micro injector until the droplet diameter is 0.27 millimeters.Hold the one cell stage embryo with tweezers and control the needle with the micro manipulator to inject 10 nanoliters of 50 mM 5 EU.Transfer the injected embryos into a new glass dish containing Ficoll solution and incubate for one hour. Remove the Ficoll solution and add 0.1 XMMR solution. Visually inspect embryos during development and remove any abnormal embryos if necessary.Transfer 10 embryos at the desired developmental stage into a 1.5 milliliter micro centrifuge tube. Remove any residual medium from the tube, snap freeze the embryos in liquid nitrogen, and store in a minus 80 degrees Celsius freezer. Then extract total RNA from the embryos, and measure its concentration using commercial kits.To make a 50 microliter click reaction, mix the components shown in a 1.5 milliliter micro centrifuge tube. Incubate the reaction mix on a nutator at room temperature for 30 minutes. Now add glycogen, ammonium acetate and chilled ethanol to each reaction mix.Mix well by pipetting five times. Incubate overnight in a minus 80 degrees Celsius freezer. After overnight incubation, centrifuge at 13, 000 G for 20 minutes at four degrees Celsius, then remove the supernatant without disturbing the pellet.Wash the pellet twice with 700 microliters of 75%ethanol. After centrifuging and discarding the supernatant, air dry the pellet at room temperature for five to 10 minutes and resuspend the pellet in 10 microliters of RNase-free water. Dispense five microliters of streptavidin-coated magnetic beads into a new 1.5 milliliter tube.Add nine microliters of wash buffer 2 to the beads and mix well. Place the tube on a magnetic rack for two minutes. Remove the supernatant and repeat the washes two more times.Then resuspend the beads in five microliters of wash buffer 2. Prepare a master mix in a 1.5 microliter microcentrifuge tube. Add 15 microliters of the master mix to each tube containing the RNA sample.Place the tube on a heat block and incubate it at 69 degrees Celsius for five minutes. Afterward, put the tube on ice. Then add five microliters of pre-washed beads to each RNA sample, and incubate on a nutator at room temperature for 30 minutes.Wash the bead RNA complexes five times with 50 microliters wash buffer 1, followed by five washes with 50 microliters wash buffer 2. Resuspend the beads in five microliters wash buffer 2. Use the beads containing the nascent EU RNAs directly for complementary deoxyribonucleic acid synthesis and library preparation.Increasing numbers of differentially activated genes were detected during zygotic genome activation when the nascent transcriptome at six hours post-fertilization was compared with that at five hours post-fertilization, with further increases observed at seven hours post-fertilization, eight hours post-fertilization, and nine hours post-fertilization. Zygotic genes were detected at higher levels, and at earlier times in the nascent EU RNA sequencing data compared with the total RNA sequencing data. Careful purification of metabolically labeled nascent RNAs is the most critical factor for ensuring sensitivity and specificity in this protocol.EU labeled nascent RNAs produced using this protocol can be further analyzed biochemically in vitro. This approach enables future studies to investigate genome activation patterns, tissue specific gene expression, and regulatory mechanisms as single cell resolution.

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Metabolic Labeling of the Nascent Transcriptome in Xenopus Early Embryogenesis

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