Fig. 3: New RNA profiling after perturbing K562 cells with different HDAC inhibitors.

a Illustration of the mini-bulk strategy for large-scale new RNA profiling after chemical compound perturbations. b Boxplots showing the signal-to-noise ratios (4sU induced conversions divided by sequencing errors) in experiments where K562 cells were exposed to 4sU alone (2 h; n = 12), 4sU combined with the transcriptional inhibitor Actinomycin D (ActD) (2 h, n = 8) and untreated negative control cells (no 4sU control, n = 12). Boxplots show median, boundaries (first and third quartile), and whiskers denote 1.5 times the interquartile range of the box. c Scatter plot showing the two first principal components (PC1 and PC2) for the panel of 28 HDAC inhibitors. For each compound, the mean of each biological replicate is shown, and each compound is colored according to inhibitor class³⁴. d Heatmap showing the union of genes that were significantly up- or down-regulated at the new RNA level when comparing each HDAC inhibitor against DMSO-treated control cells. The compounds (columns) are ordered by their PC1 score. For genes (rows), the average difference against DMSO-treated control cells is shown (log2) across three biological replicates per compound. Genes were clustered using hierarchical clustering into four main clusters (C1 – C4). e, f Volcano plots showing factors with enriched ChIP-seq binding in promoters of specific gene clusters. In e, cluster C1 is compared to the union of the genes in clusters C2 and C3; in f, cluster C3 is compared to the union of genes in clusters C1 and C2. e, f Enrichments were evaluated using Fisher’s exact test (two-sided), with the resulting P values adjusted using the Benjamini-Hochberg procedure. g Cumulative distributions of HDAC ChIP-seq reads in promoters of genes identified as significantly differentially expressed after SAHA exposure (5 μM). Comparisons are shown for new RNA analysis after 1 or 3 h of SAHA exposure, and standard total RNA-seq analysis after 6 and 24 h of SAHA treatment. Source data are provided as a Source data file.