Metabolic Labeling of the Nascent Transcriptome in Xenopus Early Embryogenesis

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.