Nascent transcriptome of Xenopus laevis embryos at mid-blastula transition (MBT) stages

To validate that EU-RNA imaging provides a direct readout of wide-spread zygotic transcription, we sought to identify the nascent transcriptome using RNA-seq. We micoinjected 5-ethynyl uridine (EU) into 1-cell Xenopus embryos, isolated total RNA from embryos at mid-blastula transition (MBT), biotinylated EU-RNA and purified it to generate cDNA libraries for sequencing. Total RNA from normal embyros were used as control. We found over 25,000 nascent genes in this EU-RNA dataset at mid-ZGA. The nascent transcriptome dataset captures nearly 90% of the transcripts present in the whole-embryo dataset and with similar or better read-depth, indicating that our EU-RNA imaging is truly representative of wide-spread zygotic transcription. Furthermore, of the known zygotic genes that are most highly induced at MBT, we detected all of them and at much higher levels than in the whole-embryo dataset. These results show that our labeling captures the nascent zygotic transcriptome. We found ~ 4-fold higher sensitivity for nascent transcripts in the EU-RNA dataset compared to the whole embryo dataset, and as excepted we did not detect thousands of maternal-only transcripts in the nascent dataset. Together, these results suggest that EU-labeling provides a visual readout of bona fide nascent zygotic transcripts. Overall design: Duplicates for nascent EU-RNA from EU-injected embryos and total RNA from normal embryos, respectively