Nascent Transcriptome of AP and VP from Xenopus laevis Embryos at 5-9 hpf (stage 7-9)

To characterize the spatial patterns of zygtoic transcription during zygotic genome activation (ZGA) of Xenopus laevis embryos, we microinjected 5-ethynyl uridine (EU) into 1-cell stage embryos and isolated total RNAs from whole embryos at 5, 6, 7, 8 and 9 hours post-fertilization (hpf) at room temperature, respectively, covering the stages of pre-ZGA to widespread ZGA (stage 7-9). The animal pole (AP, ~ 1/3 at the top region of embryo) and the vegetal pole (VP,~ 1/3 at the bottom region of embryo) regions were dissected and used for total RNA isolation. To purify nascent transcripts, total RNAs were biotinylated using disulfide biotin azide via click reaction and biotinylated nascent transcripts were purified using streptavidin beads. Libraries were constructed from using the nascent transcripts. All libraries were sequenced on illumina NextSeq 500. Overall design: Two experiments were conducted. In the first experiment, the AP and VP regions from three clutches of embryos at 5-9 hpf (three biological replicates) were used for sequecing the nascent transcripts. In the second experiment, the AP and VP regions from one clutch of embryos at 6-9 hpf (one biological replicate, two technical replicates) were used for sequencing the nascent transcripts.