Extended Data Fig. 4: Analyses of bulk large genes.

A: GRO-seq, RT and URIs along all genes over 300 kb and flanking regions (as in Fig. 3a). Bulk genes are divided into 4 groups according to their transcription levels: high (1st group, n = 119), medium (2nd group, n = 203 and 3rd group, n = 377) very low (4th group, n = 83). Genes within each group (and those hosting a SDR) are presented according to decreasing transcription levels. B-D: Boxplot comparison of URIs along large genes and flanking regions. Bounds of box: 25th and 75th percentiles; centre line: median; lower (upper) whisker: lower (upper) bound - (+) 1.5 x Interquartile range (IQR). Statistical significance was set to P≤0.05, the corresponding p values are indicated in the plots. B: Average Aph-induced URIs for genes (coloured boxes) and their flanking regions (±100 kb) (grey boxes). P values relative to the differences in URI between gene bodies and flanking regions have been calculated using one-sided Wilcoxon matched-pairs signed rank test (adjusted with FDR) with an alternative hypothesis of the gene bodies to have a lower Aph-URI compared to the flanking regions. P values for comparison of URIs between gene bodies and flanking regions are shown in black and have been calculated using two-sided Wilcoxon rank sum test and adjusted with FDR. C: Average URIs in Aph- and ARO-treated cells. Left panel: over whole gene, right panel: over later-replicated region (S50 ≥ RTmedian) of each gene. RTmedian: median replication timing for each gene. P values have been calculated as in B with alternative hypothesis of gene to have a lower URI in Aph- compared to the ARO-treated cells. D: URIs in Aph- and ARO-treated cells across genomic regions of different RT categories, defined accordingly to their S50 in NT cells; Early (0.1 ≤ S50 < 0.33, n = 18128), Mid (0.33 ≤ S50 < 0.66, n = 21319) and Late (0.66 ≤ S50 ≤ 0.9, n = 13177). Bins with extreme S50 values (S50 < 0.1 or S50 > 0.9) were excluded from this analysis. P-values have been calculated using two-sided Wilcoxon matched-pairs signed rank test. Only the late-replicating regions displayed a significant rescue.