Fig. 1: Revealing direct effects of SAHA by single-cell new RNA-sequencing. | Nature Communications

Fig. 1: Revealing direct effects of SAHA by single-cell new RNA-sequencing.

From: Deciphering direct transcriptional effects of epigenetic compounds through large-scale new RNA profiling

Fig. 1: Revealing direct effects of SAHA by single-cell new RNA-sequencing.The alternative text for this image may have been generated using AI.

a Illustration of how direct transcriptional effects of perturbations manifest rapidly, followed by propagation of indirect effects. b Illustrating 4sU incorporation into newly transcribed RNA, whereas pre-existing RNA remains unlabeled. Alkylation of 4sU bases results in the misincorporation of guanines (instead of adenines) during reverse transcription. The 4sU-induced mismatches are detected in the RNA-seq reads to distinguish newly transcribed (new RNA) from pre-existing RNA. c Experimental overview of NASC-seq2 experiment in K562. Cells were treated with SAHA and 4sU for 30 min or 1 h, and compared to control cells exposed to 4sU only or neither compound. d Violin plots summarizing the fractions of new RNA per cell, separately for each condition. e UMAP visualization of single-cell expression profiles, colored by experimental condition. Left: analysis of total (new + pre-existing) RNA per cell; right: analysis of new RNA per cell. f Bar plots showing the number of significant genes detected after 30 min and 1 h of treatment, stratified by up- and down-regulation, and by total and new RNA analysis. g Scatter plot showing the effect of SAHA treatment for 30 min (x-axis) and 1 h (y-axis), based  on total RNA. h Scatter plot showing the effect of SAHA treatment for 30 min (x-axis) and 1 h (y-axis), based on new RNA. i Volcano plot showing factors with enriched ChIP-seq binding in promoters of significantly differentially expressed genes based on total RNA after 1 h SAHA treatment. j Volcano plot showing factors with enriched ChIP-seq binding in promoters of significantly differentially expressed genes based on new RNA after 1 h SAHA treatment. i, j Enrichments were evaluated using Fisher’s exact test (two-sided), with the resulting P values adjusted using the Benjamini-Hochberg procedure. k Enrichment of histone marks near SAHA-responsive genes (1 h new RNA), based on ChIP-seq data from untreated K562 cells. Color indicates genomic region relative to gene, shape indicates significance (with or without multiple testing correction), and the x-axis the nominal P-values (Fishers exact test, two-sided). Source data are provided as a Source data file.

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