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. Author manuscript; available in PMC: 2025 Jun 20.
Published in final edited form as: Methods Mol Biol. 2025;2923:143–162. doi: 10.1007/978-1-0716-4522-2_9

Quantifying Nascent Transcription in Early Embryogenesis

Hui Chen 1
PMCID: PMC12181038  NIHMSID: NIHMS2087057  PMID: 40418448

Abstract

The early embryonic genome exists in a dormant state following fertilization, and it then subsequently undergoes broad activation of zygotic transcription in early stages of development. A major challenge is detection of newly made zygotic transcripts and determination of their activation onset in the presence of large and predominantly maternal pool of RNAs. Here we describe a detailed method to measure zygotic transcription during zygotic genome activation (ZGA) of Xenopus early embryos using metabolic labeling of nascent transcripts with 5-ethynyl-uridine (5-EU) followed by purifying EU-RNAs and sequencing them (EU-RNA-seq). This method is highly sensitive in detecting early zygotic transcripts that are not detected by total RNA-seq and determines the actual onset time of transcriptional activation for zygotic genes. The method is applicable to a wide variety of embryonic model systems and has already afforded novel insights into gene regulation in early embryogenesis.

Keywords: nascent transcription, zygotic genome activation, early embryogenesis, 5-ethynyl uridine (5-EU), RNA sequencing (RNA-seq), transcriptome

1. Introduction

Immediately following fertilization, embryogenesis is governing by maternal factors deposited in the egg, and no or little nuclear transcription occurs. After a defined period of development, the embryonic genome initiates transcription of zygotic genes that are essential for embryonic survival, cell fate specification and patterning at later stages (Jukam, Shariati, & Skotheim, 2017; Lee, Bonneau, & Giraldez, 2014; Vastenhouw, Cao, & Lipshitz, 2019). Zygotic genome activation (ZGA) during the maternal-to-zygotic transition is highly conserved in all specie s of metazoans, and the timing of ZGA is tightly controlled with in a species, although it varies between different species (Jukam et al., 2017; Lee et al., 2014; Vastenhouw et al., 2019). Further, the dysregulation of ZGA can have severe consequences in development, ranging from embryonic death to developmental defects. Thus, understanding how the early embryonic genome is activated for zygotic transcription is fundamentally important in revealing novel principles of development, which is vital for developing new strategies for treating developmental anomalies such as infertility and miscarriage, as well as developmental delays and congenital disorders.

To understand ZGA it is essential to determine the activation landscape of genes upon the onset of large-scale transcription. RNA-sequencing (RNA-seq) has been instrumental in identifying the ZGA genes and determining the kinetics of gene activation, as demonstrated by profiling of the transcriptome at different stages of development on various model organisms (Gao et al., 2018; Gentsch, Owens, & Smith, 2019; Liu et al., 2019; White et al., 2017). However, since transcripts in the early embryo is predominated by maternal RNAs, a major challenge is determining the true composition of nascent gene activation when ZGA initiates and their onset time, particularly for genes with modest expression. A recent strategy to overcome this challenge is metabolically labeling nascent RNAs using uridine analogues, such as 4-thio-uridine (4s-U) (Gentsch et al., 2019; Heyn et al., 2014) and 5-ethynyl-uridine (5-EU) (Chan et al., 2019; Chen, Einstein, Little, & Good, 2019; Chen & Good, 2022; Kwasnieski, Orr-Weaver, & Bartel, 2019), and physically isolating the newly made RNAs to selectively sequenced them. Most strikingly, compared with conventional RNA-seq, sequencing the nascent transcripts provides much higher sensitivity in detecting new genes that undergo activation during ZGA (Chen & Good, 2022). Moreover, by comparing the level of the transcripts in the egg, such a method directly distinguishes maternal-zygotic genes, transcripts of which are present in the egg and additionally induced during ZGA, and zygotic-only genes, transcription of which exclusively initiates during ZGA (Chen & Good, 2022). These methods provide invaluable new means to study the nature of ZGA and new fundamental sights into early embryonic transcription.

Here we describe the details of measuring nascent transcripts in early embryogenesis using 5-EU incorporation intro newly expressed RNAs. We use the Xenopus embryo as a model to demonstrate the delivery of 5-EU into early embryos, purification of nascent EU-RNAs and preparation of libraries for sequencing. The following sections describe procedures for (1) preparation of embryos and microinjection of 5-EU into early embryos, (2) RNA extraction and biotinylation, (3) library preparation, (4) library quantification, (5) library pooling, preparation and sequencing, and (6) analysis of the sequencing data. The overview of the key steps are shown in Fig. 1. This method has been shown to be useful for various model organisms(Chan et al., 2019; Chen et al., 2019; Chen & Good, 2020, 2022; Kwasnieski et al., 2019) and can be adapted to new embryonic models.

Fig. 1. Schematic of profiling nascent transcriptome in early embryogenesis.

Fig. 1

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(a) The fertilized Xenopus egg is microinjected with 5-ethynyl-uridine (5-EU) and the embryo develops into the blastula to mid-blastula stages when ZGA occurs. (b) The whole embryos or eggs are homogenized and the total RNAs are extracted from the embryonic lysates, including both the EU-labelled nascent RNAs (orange) and the non-nascent RNAs (maternal RNA, cyan). (c) The RNAs are conjugated with biotin azide (red) via click reaction. (d) The biotinylated nascent EU-RNAs are purified from total RNAs using streptavidin-coated beads and a magnet separator. (e-g) The nascent EU-RNAs are used for preparing libraries and RNA-seq, followed by data analysis.

2. Materials

2.1. Xenopus Embryo Preparation and Microinjection

  1. Xenopus laevis embryos: Obtained by in vitro fertilization. All animal experiments should be performed according to animal protocols approved by Institutional Animal Care and Use Committee (IACUC).

  2. 20× MMR: 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.

  3. Dejelly solution: 2% (w/v) L-cysteine in 0.1× MMR, pH adjusted to 7.8 with NaOH.

  4. Fisherbrand™ Disposable Graduated Transfer Pipettes.

  5. 3% Ficoll® 400 in 0.5× MMR.

  6. 50 mM 5-ethynyl uridine (5-EU).

2.2. RNA Extraction and Biotinylation

  1. RNase-free water.

  2. RNaseZap™ RNase Decontamination Solution.

  3. RNeasy Mini Purification Kit.

  4. DNase I recombinant, RNase-free.

  5. Qubit™ RNA HS Binding Kit.

  6. Qubit™ Flex Fluorometer.

  7. Click-iT™ Nascent RNA Capture Kit.

  8. RNaseOUT™ Recombinant Ribonuclease Inhibitor.

  9. 1 M Tris: pH adjusted to 8.5 with HCl.

  10. 1 M CuSO4: Add 1.6 g CuSO4 in 10 ml RNase-free water. Mix well.

  11. Biotin azide.

  12. Disulfide biotin azide.

  13. THPTA.

  14. 1 M Ascorbic acid: Add 1.76 g ascorbic acid in 10 ml RNase-free water. Mix well.

  15. Glycogen.

  16. 7.5 M ammonium acetate.

  17. 100% ethanol, 200 proof.

2.3. Library Preparation

  1. Universal RNA-Seq with NuQuant kit (TECAN)

  2. C1000 Touch Thermal Cycler.

  3. S220 Focused-ultrasonicator.

  4. Agilent High Sensitivity DNA Kit.

  5. 2100 Bioanalyzer Instrument.

  6. SPRIselect beads.

  7. microTUBE AFA Fiber Pre-Slit Snap-Cap (130 μl).

  8. 0.2 ml TempAssure PCR 8-tube strips with separate 8-cap (dome top) strips.

  9. 0.2 mL PCR Strip Magnetic Separator.

2.4. Library Quantification

  1. QuantStudio™ 3 Real-Time PCR System.

  2. MicroAmp™ Fast Optical 96 Well Reaction Plate, 0.1mL.

  3. Microamp™ Optical Adhesive Film.

  4. NEBNext® Library Quant Kit for Illumina®.

2.5. Library Pooling and Sequencing

  1. 1 N NaOH.

  2. 200 mM Tris, pH adjusted to 7.0 with HCl.

  3. Illumina Nextseq 500 or 550 sequencer.

  4. Illumina Nextseq 500/550 High Output kit v2.5 (75 Cycles).

  5. Kimwipes® disposable wipers.

3. Methods

3.1. Embryo Preparation and Microinjection of 5-EU into Early Embryos

3.1.1. Overview

In the following section, Xenopus embryos are used as an example for metabolic labeling of nascent RNA transcription by microinjection of 5-EU into the early embryos. It is important that clutches from multiple individual frogs should be used for measuring nascent transcripts from biological replicates (at least two). Note that 5-EU can also be administered by simply incubating embryos with 5-EU solution for other embryos such as those from zebrafish.

3.1.2. Embryo preparation

To obtain Xenopus embryos, follow a standard protocol to perform in vitro fertilization (IVF) for eggs from at least two individual frogs (Sive, Grainger, & Harland, 2007). Note that only embryos with an IVF efficiency at > 90% should be used. Dejelly embryos at ~ 30 minutes post-fertilization. Keep embryos in 0.1× Marc's modified ringers (MMR) until further use. Sort embryos if necessary. Right before microinjection, using a plastic transfer pipette to transfer (~ 50) embryos one time to a glass chamber containing 3% Ficoll/0.5× MMR.

3.1.3. 5-EU microinjection:

Briefly, fill a glass needle with 50 mM 5-ethynyl uridine (EU) and microinject 10 nl into the 1-cell Xenopus embryos to reach a final concentration of 0.5 mM EU in the embryo (see Note 1). The number of embryos to be injected depends on conditions such as the number of biological replicates and the number of time points or stages for collecting the embryos. Typically, about twenty embryos per condition are sufficient for RNA preparation. About 1 hour after microinjection, switch the embryos to and keep them in 0.1× MMR until further use. Constantly monitor the embryos if they develop normally and sort the embryos if necessary.

3.2. RNA Extraction and Biotinylation

Before RNA isolation, prepare all solutions and reagents using RNase-free water. Wipe the surface of the bench, pipettes and centrifuge with RNaseZap™ RNase Decontamination Solution to inactivate RNase.

3.2.1. Collect embryos

Transfer 20 EU-injected embryos into a 1.5 ml microcentrifuge tube at each desired time point, e.g., every 1 hour from 5-9 hours post fertilization (hpf) (see Note 2). Remove the residual medium as much as possible using a transfer pipette, and snap freeze the embryos in liquid nitrogen. After completing the collection of all replicates and time points, keep the embryos in a liquid nitrogen tank for long-term storage or a −80 °C freezer for short-term storage (see Note 3).

3.2.2. RNA extraction

Use standard methods for isolating RNAs. Here we use the RNeasy Mini Purification Kit, which works well on the yolk-rich embryos. Follow the instructions provided by the kit. The following procedures are slightly modified from the kit manual.

  1. Remove embryos from the liquid nitrogen or the −80 °C storage and place them on ice.

  2. Add 700 μl of Buffer RLT in each sample and homogenize embryos by pipetting up and down until all embryos completely dissolve. Keep samples on ice for additional 20 min and mix again by pipetting (see Note 4).

  3. Add 700 μl of 70% ethanol, mix well and transfer 700 μl of the mixture into the RNeasy spin column. Spin the column at 13,000 rpm for 1 min and remove the flowthrough.

  4. Repeat the above step for the rest of mixture.

  5. Add 350 μl of Buffer RW1 and spin the column at 13,000 rpm for 1 min.

  6. Add 80 μl of DNase I (RNase-free) to the membrane of the column and incubate for 15 min at room temperature.

  7. Add 350 μl of Buffer RW1 and spin the column at 13,000 rpm for 1 min.

  8. Add 500 μl of Buffer RPE and spin the column at 13,000 rpm for 1 min.

  9. Repeat the above step once.

  10. Dry the column by spinning the column again at 13,000 rpm for 2 min.

  11. Transfer the column to a new tube and add 35 μl of RNase-free water to the membrane of the column. Spin the column at 13,000 rpm for 1 min.

  12. Collect the elute RNAs and keep them on ice.

3.2.3. Measure RNA concentration

Use Qubit™ RNA HS Binding Kit and the Qubit™ Flex Fluorometer. Follow the instructions provided by the kit. The following steps are slightly modified.

  1. Set up 0.2 ml 8-tube strips: use 2 tubes for the standards and 1 tube for each sample.

  2. Prepare the working solution: Dilute the Qubit™ RNA Reagent (dye) at 1:200 in the Qubit™ RNA Buffer. Budget 200 μl for each standard or sample.

  3. Dilute standards and samples in the working solution: For standards, add 10 μl into 190 μl of the working solution, and for samples, add 1 μl into 199 μl of the working solution.

  4. Mix well and put the tube strip in the Qubit™ Flex Fluorometer. Select RNA Assay, calibrate with standards, and then read the samples. Record the concentrations of each sample.

3.2.4. Biotinylate RNA:

Use the Click-iT™ Nascent RNA Capture. Follow the instructions provided by the kit; also see (Palozola, Donahue, & Zaret, 2021). Alternative reactions using biotin azide or disulfide biotin azide can also be performed to biotinylate RNAs, as described below.

  1. For each sample, prepare a 50 μl click reaction mix in a 1.5 ml microcentrifuge tube, which contains total RNA (2.5-10 μg, depending on the yield), 1× Click-iT™ EU Buffer, 2 mM CuSO4, 0.25-1 mM biotin azide (depending on RNA amount used), 10 mM additive 1, and 12 mM additive 2 (see Note 5). Incubate the reaction mix at room temperature on a nutator for 30 min.

  2. Alternative approach: For each reaction, include total RNA, 50 mM Hepes (pH 7.5), 2 mM disulfide biotin azide, 1.25 mM CuSO4/THPTA mix and 10 mM ascorbic acid and incubate the reaction for 1 h at room temperature.

3.2.5. Precipitate RNA

Follow the instructions provided by the Click-iT™ Nascent RNA Capture Kit. Note that some reagents used in these steps are not included in the kit.

  1. In each reaction tube, add 1 μl of glycogen, 50 μl of 7.5 M ammonium acetate, and 700 μl of chilled 100% ethanol. Mix well by gently inverting the tube for five times. Incubate the tubes overnight in a −80 °C freezer.

  2. Centrifuge the tube at 13,000 g for 20 min at 4 °C. Remove the supernatant.

  3. Add 700 μl of 75% ethanol to the pellet, mix gently and centrifuge the tube at 13,000 g for 5 min at 4 °C. Remove the supernatant.

  4. Repeat once the above step.

  5. Air dry the pellet for 5-10 min at room temperature.

  6. Resuspend the pellet in 10 μl RNase-free water.

    Optional: in this step, spike-in control RNAs can be added.

3.2.6. Purify the biotinylated EU-RNA

Follow the instructions provided by the Click-iT™ Nascent RNA Capture Kit. The following steps are slightly modified.

  • 1)

    Prepare beads: For each sample, dispense 5 μl of Dynabeads® MyOne™ Streptavidin T1 into empty 1.5-ml microcentrifuge tubes. Centrifuge briefly and gently remove the supernatant without disturbing the beads. Wash the beads 3 times with 9 μl of the Wash Buffer 2. Remove the supernatant. Resuspend beads in 5 μl of Wash Buffer 2.

  • 2)

    Prepare reaction: Dilute the RNaseOUT™ Recombinant Ribonuclease Inhibitor 1:10 in RNase-free water before use. For each reaction, set up the 0.2 ml tubes and prepare a master mix as follows (15 μl for each tube): 12.5 μl of Click-iT RNA binding buffer (Component G), 0.2 μl of pre-diluted RNaseOUT™, and 2.3 μl of RNase-free water. Incubate the tubes on a heat block at 69 °C for 5 min. Put the tubes on ice.

  • 5)

    Add 5 μl of pre-washed beads into each reaction. Incubate at room temperature for 30 min on a nutator.

  • 6)

    Wash beads: 5 times with 50 μl of Wash Buffer 1, followed by 5 times with 50 μl of Wash Buffer 2. For each wash, gently mix the beads with the wash buffer and put the tubes on a magnetic separator to immobilize the beads. Remove the supernatant without disturbing the beads.

  • 7)

    Resuspend beads in 5 μl of Wash Buffer 2. These beads are ready for direct cDNA synthesis and library preparation.

    Note: The total RNA and the flowthrough from the nascent EU-RNA pulldown can also be used for preparing libraries for RNA-seq, which can be used to compare the expression level from various populations of RNA (Fig. 2).

Fig. 2. Nascent EU-RNA-seq demonstrates high sensitivity in measuring zygotic transcripts.

Fig. 2

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(a) Schematic of using 5-EU injected Xenopus embryos for different RNA-seq strategies. The total RNAs are measured by conventional RNA-seq, or they can be separated into nascent EU-RNA and flowthrough (maternal RNA) for RNA-seq respectively. (b) Expression of an zygotic gene (bix1.1.L) measured by RNA-seq for different types of RNAs. For each type, dashed lines indicate two replicates, and the thick lines indicate the average. (c) Genome browser viewer of an example gene (bix1.1.L) measured by different RNA-seq strategies (Chen & Good, 2022). This zygotic gene is detected earlier with higher level by nascent EU-RNA-seq than total RNA-seq, but not detected by flowthrough RNA-seq.

3.3. Library Preparation

To prepare libraries, use commercially available kits and follow instructions provided by the kit. Here we describe the procedures using the Universal RNA-Seq with NuQuant kit, with modifications from its manual. This kit uses the custom designed AnyDeplete Probe Mix for Xenopus laevis to deplete ribosomal RNAs before making the cDNA libraries. Before each step in the following section, remove the related reagents from the −20 °C storage and keep them on ice until they are completely thawed before use. Briefly spin the tubes before use. For nuclease-free water and Agencourt beads used in purifying DNA wherever applicable, make sure they reach room temperature before use.

3.3.1. First Strand cDNA Synthesis

Add 2 μl of First Strand Primer Mix to each sample tube that contains 5 μl of RNA on beads prepared from above. Mix well by pipetting. Incubate at 65 °C for 1 min in the pre-warmed C1000 thermal cycler (Bio-Rad). Remove the tubes from the thermal cycler and put them on ice. Add 2.5 μl of First Strand Master Mix and 0.5 μl of First Strand Enzyme Mix into each sample tube. Mix well by pipetting. Incubate at 25 °C for 5 min, 40 °C for 5 min, 70 °C for 15 min, and hold at 4 °C in the pre-warmed thermal cycler. Remove the tubes from the thermal cycler, briefly spin the tubes and place them on ice.

3.3.2. Second Strand cDNA Synthesis

Add 63 μl of Second Strand Buffer Mix and 2 μl of Second Strand Enzyme Mix in each sample tube from above. Mix well by pipetting. Incubate at 60 °C for 60 min in the pre-warmed thermal cycler. Remove the tubes from the thermal cycler, briefly spin the tubes and place them on ice. Add 45 μl of Second Strand Stop Buffer to each sample tube for a total of 120 μl. Mix well by pipetting. Continue immediately to the following steps for cDNA fragmentation or store the samples at −20 °C.

3.3.3. cDNA Fragmentation

Place microTUBE tubes on a holder. Add the 120 μl DNA sample into each microTUBE, and top with Nuclease-free water followed by capping the tube. Keep the tubes on ice. Sonicate the tubes inside the Covaris S220 sonicator following the instructions provided by the manufacturer (see Note 6). Transfer 100 μl of the fragmented cDNA from the Covaris tube into 0.2 ml tubes. Place the tubes at room temperature.

3.3.4. cDNA Concentration

Prepare the 70% ethanol wash solution, and make sure the nuclease-free water and Agencourt beads reach room temperature before use. Invert the tubes to ensure the beads are fully resuspended before adding to the sample. Add 180 μl (1.8 volumes) of bead suspension to the 100 μl fragmented cDNA. Mix well by pipetting. Equally split each sample into two 140 μl aliquots and incubate at room temperature for 10 minutes. Transfer the sample tubes to the magnet and keep for 5 minutes until the solution is clear. Carefully remove the binding buffer without disturbing the beads on the wall of tube. With the sample tubes still on the magnet, add 200 μl of freshly prepared 70% ethanol and allow to stand for 30 seconds. Remove the 70% ethanol wash using a pipette. Repeat the wash with 70% ethanol once more. Air-dry the beads on the magnet for 10 minutes or until all ethanol has evaporated. Remove the tubes from the magnet. Add 11 μl of Nuclease-free water to the dried beads in one of the two tubes for each sample. Resuspend the beads by pipetting. Transfer the resuspended beads to the second tube. Resuspend the beads by pipetting and incubate at room temperature for 3 minutes. Transfer the tubes to the magnet and keep for 3 minutes until the solution is clear. Transfer 10 μl of the eluates to new 0.2 ml tubes without disturbing the beads. Keep them on ice.

3.3.5. End Repair

Add 4 μl of End Repair Buffer Mix, 0.5 μl of End Repair Enzyme Mix, and 0.5 μl of End Repair Enhancer in each sample tube above. Mix well by pipetting. Incubate at 25 °C for 30 min, 70 °C for 10 min and hold at 4 °C in the pre-warmed thermal cycler. Remove the tubes from the thermal cycler, briefly spin the tubes and place them on ice.

3.3.6. Adaptor Ligation

Add 4.5 μl of Nuclease-free water, 6 μl of Ligation Buffer Mix, 1.5 μl of Ligation Enzyme Mix, and 3 μl of individual adaptor mix from the Adaptor Plate to each sample tube from above. Mix well by pipetting. Incubate at 25 °C for 30 min and hold at 4 °C in the pre-warmed thermal cycler. Remove the tubes from the thermal cycler, briefly spin the tubes and place them on ice.

3.3.7. Strand Selection

Add 69 μl of Strand Selection Buffer Mix 1 and 1 μl of Strand Selection Enzyme Mix 1. Mix well by pipetting. Incubate at 72 °C for 10 min and hold at 4 °C in the pre-warmed thermal cycler. Remove the tubes from the thermal cycler, briefly spin the tubes and place them on ice.

3.3.8. Strand Selection Purification

Make sure nuclease-free water and Agencourt beads reach room temperature before use. Invert the tubes to ensure the beads are fully resuspended before adding to the sample. Add 80 μl (0.8 volumes) of the Agencourt beads suspension to the Strand Selection reaction product. Mix well by pipetting. Incubate at room temperature for 10 minutes. Transfer the sample tubes to the magnet and keep for 5 minutes until the solution is clear. Carefully remove165 μl of the binding buffer without disturbing the beads on the wall of tube. Leave some of the volume behind minimizes bead loss. With the sample tubes still on the magnet, add 200 μl of freshly prepared 70% ethanol and allow to stand for 30 seconds. Remove the 70% ethanol wash using a pipette. Repeat the wash with 70% ethanol once more. Air-dry the beads on the magnet for 10 minutes or until all ethanol has evaporated. Remove the tubes from the magnet. Add 16 μl of Nuclease-free water to the dried beads. Resuspend the beads by pipetting and incubate at room temperature for 3 minutes. Transfer the tubes to the magnet and keep for 3 minutes until the solution is clear. Transfer 15 μl of the eluates to new 0.2 ml tubes without disturbing the beads. Keep them on ice.

3.3.9. Probe Binding

Add 5 μl of AnyDeplete Buffer Mix, 0.5 μl of Strand Selection Enzyme Mix II, 4 μl of AnyDeplete Probe Mix, and 0.5 μl of AnyDeplete Enzyme Mix I to each sample tube from above. Mix well by pipetting. Incubate at 37 °C for 10 min, 95 °C for 2 min, 50 °C for 30 s, 65 °C for 5 min, and hold at 4 °C in the pre-warmed thermal cycler. Remove the tubes from the thermal cycler, briefly spin the tubes and place them on ice.

3.3.10. Targeted Depletion

Add 16 μl of nuclease-free water, 5 μl of AnyDeplete Buffer Mix, and 4 μl of AnyDeplete Enzyme Mix II to each sample tube from above. Mix well by pipetting. Incubate at 60 °C for 30 min, 95 °C for 5 min, and hold at 4 °C in the pre-warmed thermal cycler. Remove the tubes from the thermal cycler, briefly spin the tubes and place them on ice.

3.3.11. Library Amplification

Add 31.5 μl of nuclease-free water, 10 μl of Amplification Reagent I, 8 μl of Amplification Reagent II, and 0.5 μl of Amplification Enzyme Mix to each sample tube from above. Mix well by pipetting. Incubate at 95 °C for 2 min, 2 cycles of 95 °C for 30 s and 60 °C for 90 s, 18 cycles of 95 °C for 30 s and 65 °C for 90 s, 65 °C for 5 min and hold at 4 °C in the pre-warmed thermal cycler. Note that the number of PCR cycles for each individual sample can vary and the optimal cycles can be predetermined by using a small portion of sample. Remove the tubes from the thermal cycler, briefly spin the tubes and place them on ice.

3.3.12. Amplified Library Purification

Prepare the 70% ethanol wash solution, and make sure the nuclease-free water and Agencourt beads reach room temperature before use. Invert the tubes to ensure the beads are fully resuspended before adding to the sample. Add 100 μl (1 volume) of bead suspension to the 100 μl Library Amplification reaction product. Mix well by pipetting. Incubate at room temperature for 10 minutes. Transfer the sample tubes to the magnet and keep for 5 minutes until the solution is clear. Carefully remove the binding buffer without disturbing the beads on the wall of tube. Remove the sample tubes from the magnet, add 200 μl of freshly prepared 70% ethanol and mix thoroughly by pipetting. Transfer the tubes to the magnet and allow to stand for 5 minutes until the solution is clear. Remove the 70% ethanol wash using a pipette. Repeat the wash with 70% ethanol once more. Air-dry the beads on the magnet for 10 minutes or until all ethanol has evaporated. Remove the tubes from the magnet. Add 26 μl of nuclease-free water to the dried beads. Resuspend the beads by pipetting and incubate at room temperature for 3 minutes. Transfer the tubes to the magnet and keep for 3 minutes until the solution is clear. Transfer 25 μl of the eluates to new 0.2 ml tubes without disturbing the beads. Keep them on ice.

3.3.13. Quality Check (Optional)

The quality of the libraries can be assessed using the Agilent High Sensitivity DNA Kit in the 2100 Bioanalyzer Instrument. Follow the instructions provided by the kit. The ideal size range of the libraries is ~100-500 bp, with a peak at ~300 bp.

3.3.14. Size Selection (Optional)

Specific size range of fragments in the libraries can be selected using SPRIselect beads. Follow the instruction provided by the manufacturer.

3.4. Library Quantification

The concentration of libraries prepared from above can be quantified using the NEBNext® Library Quant Kit for Illumina® kit. Follow the instruction manual provided by the kit. The following procedures are adapted from the manual with slight modifications.

3.4.1. Prepare reagents

Thaw the kit components at room temperature. Mix well and briefly spin the tube. Add 100 μl of the NEBNext® Library Quant Primer Mix and 20 μl of ROX (low) to the tube of NEBNext® Library Quant Master Mix (1.5 ml). Mix well by pipetting. Prepare the 1× NEBNext® Library Quant Dilution Buffer by diluting the 10× buffer at 1:10 in nuclease-free water.

3.4.2. Dilute Libraries

Dilute each library at 1:1,000 in the 1× NEBNext® Library Quant Dilution Buffer (1 μl of library + 999 μl of buffer). Further dilute the diluted libraries at 1:10,000 (10 μl of 1:1,000-diluted library + 90 μl of buffer) and 1:100,000 dilutions (10 μl of 1:10,000-diluted library + 90 μl of buffer) - these two dilutions will be used for quantification. Mix well before use.

3.4.3. Preform real-time quantitative PCR (qPCR)

Set up triplicates for DNA standards, diluted libraries, and no-template control (NTC) using the online tool for QuantStudio™ 3 Real-Time PCR System. For each reaction in a 96-well plate, add 16 μl of NEBNext® Library Quant Master Mix (with primers) 16 μl and 4 μl of DNA standards or library dilutions. Mix reactions well by pipetting. Cover the 96-well plate with clear plastic cover and centrifuge for 5 min at 3,000 g. Run quantitative real-time PCR in the QuantStudio™ 3 Real-Time PCR System using the following cycling conditions: 95°C for 1 min (Initial Denaturation); 35 cycles of 95°C for 15 s (Denaturation) and 63°C for 45 s (Extension).

3.4.4. Calculate library concentrations

Use the Cq values of the 0.01-10 nM DNA standards to generate a standard curve. The concentrations of libraries can be calculated from the standard curve (see Note 7). The final library concentrations can be adjusted for size by multiplying the calculated concentration with 399/library size (bp), in which the library size can be determined by Bioanalyzer.

3.5. Library Pooling and Sequencing

3.5.1. Pooling and Preparation for Sequencing.

Before pooling, set up a target final volume and concentration, e.g., 10 μl at 2 nM, which can be used directly in the following steps as described below (see Note 8). Calculate the volumes required for each library to be pooled using the concentrations determined above and mix individual libraries at an equal molar ratio. If the concentrations between libraries vary dramatically, make sure that there is enough volume for those libraries at a low concentration. After mixing the libraries, quantify the final concentration of the pooled library again using qPCR as described above. It is important that the final concentration is accurately measured.

Before sequencing, the libraries need to be denatured and diluted to a concentration optimal for sequencing. Below describe the procedures for getting the libraries ready for sequencing, using 10 μl of libraries at the starting concentration of 2 nM as an example, modified from the Denature and Dilute Libraries Guide for the NextSeq System by Illumina. Please refer to the Guide for adjusting the volumes of reagents for libraires at different concentrations.

  1. Prepare reagents:
    1. Prepare 0.2 N NaOH: To make 1 ml of 0.2 N NaOH, add 200 μl of 1N NaOH into 800 μl water and mix well (see Note 9).
    2. Prepare HT1 hybridization buffer: Remove HT1 from the −20 °C storage and thaw at room temperature. Keep at 4 °C or on ice until ready for use.

  2. Denature libraries: Add 10 μl of fresh 0.2 N NaOH into the libraries. Mix well by pipetting multiple times. Incubate for 5 min at room temperature.

  3. Neutralize libraries: Add 10 μl of 200 mM Tris, pH 7.0. Mix well.

  4. Dilute the denatured libraries to 20 pM: Add 970 μl of chilled HT1 hybridization buffer into the libraries from the last step. Mix well and keep on ice.

  5. Dilute the libraries to 1.8 pM: Mix 117 μl of the 20 pM libraires with 1183 μl of chilled HT1 hybridization buffer (see Note 10). Keep on ice. The libraries are ready for loading into a sequencer.

3.5.2. Sequencing

The libraries prepared from above can be sequenced using various sequencing kits and by several different brands of sequencers commercially available. Here we use the Illumina NextSeq 500 or 550 sequencer and Nextseq 500/550 High Output kit v2.5 (75 Cycles). Before sequencing, thaw the reagents cartridge from the sequencing kit overnight at 4 °C. In addition, one can sign up for the BaseSpace, which is an online tool provided by Illumina for planning the sequencing runs, store the sequence results, file conversion, and monitor the sequencing process.

  1. Turn on the sequencer and follow the instructions to sequentially load the components from the sequencing kit. Optional: Log into BaseSpace on the sequencer and select the planned run. If it is prompted, provide the parameters in the sequencing settings based on the kit used for preparing the libraries; for example, for paired-end sequencing of the libraries prepared by the Universal RNA-Seq with NuQuant kit above, the following parameters can be used: Read1, 42; Read2, 42; and Index1, 8 (see Note 11). Note that the Nextseq 500/550 High Output kit v2.5 (75 Cycles) can be used for up to 92 total cycles.

  2. Open the flow cell, and make sure it is intact and there is no scratch or crack on the surface. Clean the top glass surface by using 70% ethanol and Kimwipes® disposable wipers. Load the flow cell onto the sequencer by following the instruction.

  3. Load the buffer cartridge by following the instruction.

  4. Gently shake or invert the reagent cartridge several times to mix the reagents inside. Make sure that the reservoirs are completely thawed. Clean the surface of the #10 position of the reagent cartridge with a clean Kimwipes® disposable wiper and pierce it with a P-1000 pipette tip. Pipette the1.3 ml of the 1.8 pM pre-diluted library into the well of # 10. Load the reagent cartridge by following the instruction suggested by the sequencer.

  5. Press “Sequence”. The sequencer will check the system and start running sequencing. The process of sequencing can be monitored in BaseSpace if logged in. The sequencing results, including the binary base call (BCL) sequence files, will be stored in BaseSpace, and the FASTQ files will be automatically generated from the BCL files. To get ideal sequencing results, it is recommended that the optimal cluster density is at ~170-220 k/mm2.

3.6. Data analysis

The sequencing results can be analyzed using commonly used bioinformatic tools. For example, to measure the expression level of transcripts, sequences in FASTQ files can be aligned to the genome (e.g., Xenopus laevis genome build 9.2) and quantified by salmon (Patro, Duggal, Love, Irizarry, & Kingsford, 2017). Further, differential gene expression analysis can be performed using DESeq2 (Love, Huber, & Anders, 2014) (Fig. 2). Moreover, the sequences can be aligned to the genome using bowtie2 (Langmead & Salzberg, 2012), and sequence alignment map (SAM) or binary alignment map (BAM) files and bigWig files can be generated by using samtools (Li et al., 2009) and deeptools (Ramirez et al., 2016), respectively. The bigwig files can be used in the Integrative Genomics Viewer (IGV) (Robinson et al., 2011) for visualizing the peaks of expression in each gene. Alternatively, the sequences can me mapped to the genome using the STAR aligner (Dobin et al., 2013) and the generated tiled data file (tdf) files can also be used for visualization in the genome browser (Fig. 2).

The expression of nascent RNAs can be filtered using a cutoff of DESeq2 normalized reads to determine whether a gene is zygotically expressed. To further distinguish the maternal-zygotic and zygotic-only genes, the reads from embryos at ZGA stages can be compared with the unfertilized eggs or those at pre-ZGA stages (Chen & Good, 2022). The functional differences between these groups of genes can be summarized by gene ontology (GO) analysis, e.g., using clusterProfiler (Wu et al., 2021), and the enrichment of functional motifs in differentially expressed genes can be retrieved by using HOMER (Duttke, Chang, Heinz, & Benner, 2019). To determine the transcriptional activation onset time for each ZGA gene, the expression profiles can be fitted with mathematical models (Chen & Good, 2022; Jukam, Kapoor, Straight, & Skotheim, 2021). Finally, the sequencing results from different regions of early embryos can be used for discovering new spatiotemporal patterns of gene activation and uncovering the underlying mechanisms (Chen & Good, 2022).

Summary:

Measuring the nascent transcriptome in early embryogenesis is important for understanding of the functional regulation of the embryonic genome. The highly sensitive methods such as the EU-RNA-seq presented above allow us to gain better insights into early embryonic genome activation. Importantly, the method is compatible with single-cell analysis as well as spatial analysis. By integrating with other multi-omics approaches and applying them in various embryonic systems, it will continue to help reveal novel principles of early development and facilitate the development of new means of diagnosis and treatment for developmental diseases.

4. Notes

  1. For any other given embryonic system, the optimal concentration of 5-EU should be empirically determined by microinjecting 5-EU at various concentrations and determining their toxicity on embryo development. Microinjecting too much 5-EU could lead to developmental delay or arrest of the embryos, while microinjecting too little 5-EU could reduce the sensitivity of its labeling the nascent transcripts. The optimal concentration should demonstrate sensitivity in detecting nascent transcripts, e.g., nascent by single-cell EU-RNA imaging (Chen & Good, 2020), but no obvious toxic effect on early embryos.

  2. The number of embryos to be collected depends on the yield of RNA from early embryos, and thus it should be empirically determined before experiment. A typical yield of RNA from early Xenopus embryos is ~2-5 μg per embryo. Make sure that the yield of RNA is sufficient for downstream analysis in EU-RNA biotinylation.

  3. For RNA extraction, it is preferred to use freshly collected embryos or stored embryos as early as possible. Because RNAs are easily degradable, long-time storage of the embryos could result in reduction in RNA yield and compromise the sensitivity in measuring the zygotic genes that are lowly activated during ZGA.

  4. Note that the embryos can be difficult to be lysed and it could take some time for them to completely dissolve. By pipetting more frequently, prolonging the incubating time on ice or employing a tissue homogenizer, it could help facilitate the homogenization process. After lysis of embryos, the solution can be sticky. To prevent the loss of materials, one should handle the samples with extra care. Complete homogenization of the embryos without significant loss is critical for the downstream steps of RNA preparation.

  5. Follow the instructions of the kit to determine the amount of total RNA and biotin azide to be used in the click reaction. For example, for 1-5 μg of total RNA, 0.5 mM biotin azide can be used, while for 10 μg of RNA, 1 mM biotin azide can be used. The optimal amount can also be empirically determined by testing the efficiency of the click reaction using varying concentrations of samples and reagents.

  6. The sonicating parameters, including the power, duty factor, cycles per burst, and treatment time, can be different for embryos at various stages, dependent on the chromatin stages, and thus the optimal conditions should be predetermined before experiment. The ideal size range of the DNA fragments after sonication is ~100-500 bp, with a peak at ~ 300 bp.

  7. When calculating the concentrations from the standard cure, only the values fall within the range of the standards should be used. The average concentrations calculated from different dilutions should be used, which should further be corrected by the fragment sizes.

  8. Follow the instructions of the Denature and Dilute Libraries Guide for the NextSeq System to determine the recommended target volumes and concentrations that are appropriate for downstream analysis. A typical starting volume for different library concentration can be 5 μl for 4 nM, 10 μl for 2 nM, 20 μl for 1 nM, and 40 μl for 0.5 nM. The same volumes of 0.2 N NaOH should be added to libraries with respective concentrations, no more than 1 mM NaOH is in the final diluated sample.

  9. The 0.2 N NaOH solution should be freshly made right before use, which is essential for denaturing the libraries for cluster generation.

  10. The final volume and concentration of the diluted libraries is 1.3 ml at 1.8 pM. However, depending on the quality of libraries and the sequencing capacity of the kits, the final concentration can be slightly adjusted to be higher (e.g., to 2.2-2.4 pM) but should aim for an optimal cluster density of ~170-220 k/mm2 on a flow cell, which maximizes the yield of sequencing reads and data quality.

  11. For nascent transcription measurement, it is recommended to perform paired-end sequencing. Paired-end sequencing allows more accurate read alignment to the genome and facilitates the detection of spliced RNAs and novel transcripts. This could be critical for measuring the transcribed genes with different isoforms, such as differentiating Xenopus genes activated from the long (L) and short (S) subgenomes.

Acknowledgements

I would like to thank the lab of Matthew Good at the University of Pennsylvania for training on this project and National Xenopus Resource (NXR) for guidance on Xenopus. I also thank the Department of Cell and Developmental Biology, the Epigenetics Institute, and the Institute of Regenerative Medicine at the University of Pennsylvania for providing generous support. This work was supported in part by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R03HD105802).

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