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Fig. 3 Evidence for spurious transcription at hundreds of gene loci prior to MZT (A) T>C confidence score (PTC) reports the percentage of T>C conversions per 4sU-labeled transcript. Only genes that exhibit a PTC score corresponding to a true positive rate of >74.4% and false positive rate of 0% (see Figure S3A) are shown. Previously reported early transcribed genes are highlighted (red). n, number of genes. See also Table S2. (B) Venn diagrams report the overlap of genes detected above PTC cutoff in two independent biological replicates at the indicated time points (in hpf). p value is indicated (Fisher’s exact test, ∗∗∗∗p < 10−4). (C) Heatmap for all genes detected above PTC cutoff at 0.75 hpf (2-cell stage; n = 501) reports the presence of T>C reads (above PTC cutoff) in none (white), one (gray), or both (black) of two independent experiments at the indicated subsequent time points (in hpf). Genes with continuous (red) or discontinuous signal (blue) in at least one of two replicates are highlighted (left). (D) Median number of introns (left) or length of the coding sequence (CDS; in kilobases [kb]) for the indicated number of genes ranked by highest (filled circles) or lowest (open circles) PTC at 0.75 hpf. Statistically significant deviation (Wilcoxon test; ∗∗∗∗p < 10−4) from median (dashed line) and interquartile range (gray area) of all genes in the dataset is indicated. (E) Normalized enrichment scores (NES) of ATAC-seq (left; derived from Pálfy et al.9) and H3K27 acetylation chromatin immunoprecipitation sequencing (ChIP-seq) (right; derived from Bogdanović et al.25) for the indicated number of genes ranked by highest (red) or lowest (blue) PTC at 0.75 hpf. Significant enrichment (hypergeometric test with Benjamini-Hochberg correction; p < 0.05) is indicated in opaque colors.