Metabolic Labeling of the Nascent Transcriptome in Xenopus Early Embryogenesis

Summary

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

Abstract

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.

Introduction

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 formation^1,2,3,4,5,6. 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 species^1,2,3,4,5,6. 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 method^10,13 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, 1–10 µg 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.

Protocol

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

1. 20x MMR (Marc'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.
2. 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 °C until use.
3. 5-ethynyl uridine (5-EU) stock solution: To make the 50 mM stock solution, add 372.8 µL 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 µL aliquots. Store at -80 °C.

2. Microinjection of 5-EU into 1-cell embryos

1. 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.
2. 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.
3. 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 µL of 50 mM 5-EU from the top of the glass needle.
4. Assemble the needle onto a micromanipulator. Use the P10 pipette to add 10 µL of halocarbon oil on a stage micrometer and put it under a stereomicroscope.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. Store the embryos in a -80 °C freezer until further use.

3. Total RNA Extraction and Concentration Measurement

NOTE: Use a silica membrane–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.

1. Put the samples from the -80 °C freezer on ice.
2. Add 700 µL 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.
3. 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.
4. Add 700 µL of 70% ethanol to the homogenized embryos. Pipette up and down to mix well and transfer the 700 µL of the mixture into a spin column.
5. 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.
6. Add 350 µL of the Buffer RW1 and centrifuge the column at 10,000 x g for 1 min. Discard the flowthrough.
7. Carefully add 80 µL of DNase I to the center of the membrane in the column. Incubate for 15 min at room temperature.
8. Add 350 µL of Buffer RW1 and centrifuge the column at 10,000 x g for 1 min. Discard the flowthrough.
9. Add 500 µL of Buffer RPE and centrifuge the column at 10,000 x g for 1 min. Discard the flowthrough. Repeat the washing once more.
10. Dry the column by centrifuging it again at 10,000 x g for 2 min.
11. Transfer the column to a new RNase-free tube. Carefully add 35 µL of RNase-free H₂O to the center of the membrane in the column.
12. Centrifuge the column at 10,000 x g for 1 min. Collect the eluted RNA and keep on ice.
13. 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 µL for each standard or sample.
14. Set up a 0.2-mL 8-tube strip. To dilute each standard, add 10 µL to 190 µL of the working solution. To dilute each sample, add 1 µL to 199 µL of the working solution. Mix well.
15. Put the 8-tube strip in a fluorometer. Select the RNA Assay, calibrate with the standards, and read the concentrations of the samples.

4. Nascent EU-RNA Biotinylation and Purification

NOTE: A click chemistry–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.

1. RNA Biotinylation and Cleaning
   1. For each sample, mix the following components in a 1.5 mL microcentrifuge tube to make a 50-µL click reaction: total RNA (1–10 µg), 1x EU buffer, 2 mM CuSO₄, 0.25 mM biotin azide, 10 mM additive 1, and 12 mM additive 2.
   2. Incubate the reaction mix at room temperature for 30 min on a nutator.
   3. In each reaction above, add 1 µL of glycogen, 50 µL of 7.5 M ammonium acetate, and 700 µL of chilled 100% ethanol. Pipette up and down 5 times to mix them well.
   4. Put the tube in a -80 °C freezer and keep it overnight.
   5. Centrifuge the tube at 13,000 x g for 20 min at 4 °C. Carefully remove the supernatant without disturbing the pellet.
   6. Add 700 µL of 75% ethanol and mix gently to resuspend the pellet. Centrifuge the tube at 13,000 x g for 5 min at 4 °C. Carefully remove the supernatant without disturbing the pellet. Repeat the washing once more.
   7. Air dry the pellet for 5–10 min at room temperature. Resuspend the pellet in 10 µL RNase-free H₂O.
2. EU-RNA Purification
   1. To pull down the nascent EU-RNAs, for each sample, dispense 5 µL of streptavidin-coated magnetic beads in a new 1.5 mL microcentrifuge tube.
   2. Add 9 µL 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 µL of Wash Buffer 2.
   3. Prepare the following master mix in a new 1.5 mL microcentrifuge tube (15 µL for each sample): 12.5 µL of RNA binding buffer (Component G), 0.2 µL of 1:10 diluted RNase OUT, and 2.3 µL of RNase-free H₂O. Add 15 µL of the mix to each tube containing the RNA sample.
   4. Incubate for 5 min on a heat block at 69 °C. Put the tube on ice.
   5. Add 5 µL of the above pre-washed beads to each tube containing the RNA sample. Incubate for 30 min on a nutator at room temperature.
   6. Add 50 µL 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.
   7. Add 50 µL 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.
   8. Resuspend the beads in 5 µL 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).

5. RNA Library Preparation and Sequencing

1. 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.
2. 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.
3. 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.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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