Figure 1. Nascent EU-RNA-seq to characterize the composition and dynamics of ZGA with high sensitivity.
(A) Schematic of transcript composition during zygotic genome activation in early embryogenesis; from egg to late blastula. Red, transcripts of zygotic genes; Orange, transcripts of maternal-zygotic genes; Blue, transcripts of maternal genes.
(B) Schematic describing the EU-RNA-seq methodology. Nascent transcripts are metabolically labeled via 5-ethynyl uridine (5-EU) microinjected in 1-cell Xenopus embryos. Total RNAs are isolated for biotinylation via click reaction. The 5-EU labeled nascent transcripts (red) are captured by streptavidin beads; flowthrough contains maternal transcripts.
(C) Distinguishing nascent transcriptome (‘Bead’) versus maternal transcriptome (‘Flowthrough’) reads via RNA-seq from 5–9 hpf in blastula embryos. Each dot represents individual genes with rlog reads averaged from replicates quantified by DESeq2. Dashed lines: 1.5-fold threshold for enrichment.
(D) Nascent reads enrich in ‘Bead’ library versus traditional total transcriptome (‘All’) at 7 hpf from RNA-seq. Each dot represents individual genes with rlog reads averaged from replicates quantified by DESeq2. Dashed lines: 1.5-fold threshold for enrichment.
(E) Higher sensitivity for detection of zygotic expression in nascent transcriptome versus total transcriptome, at all gene expression levels.
(F) Hundreds of transcripts are uniquely detected by nascent transcriptome. Mean reads from duplicates (mean ± SE) for 240 genes detected from nascent EU-RNA (red) and total RNA (orange), respectively.
(G) Percentage of genes expressed during ZGA that are classified as zygotic-only genes (Z) and maternal-zygotic gene (MZ).
(H-I) Percentage of total library reads from transcripts of zygotic-only genes (Z) and maternal-zygotic gene (MZ) from 5–9 hpf.
(J and K) Gene ontology (GO) analysis of MZ (J) and Z (K) genes.