Extended Data Fig. 3: Outline and controls for SLAM-seq experiments.

(a) Experimental design. To validate the SLAM-seq protocol, we performed a pilot experiment for mapping responses to short-term THZ1 (2h) treatment by SLAM-seq in mES cells. SLAM-seq utilizes thymine-to-cytosine (T>C) conversion from 4-thiouridine (4sU)-labelled mRNAs to quantify the abundance of nascent RNA transcripts using 3′ end mRNA-sequencing (Quant-seq). To monitor the consistency and reproducibility of different SLAM-seq data, we inhibited transcription with THZ1 (reduces RNAPII-mediated gene transcription by inhibiting cyclin-dependent kinase 7 (CDK7)). (b) Conversion rates for each position of 4-thioU-containing SLAM-seq reads (≥ 2 T>C conversions) before or after Auxin or THZ1 treatment for 2 h. Changes in the abundance of newly synthesized mRNAs (detected in SLAM-seq based on T>C conversions). Average conversion rates (centre line) ± s.d. (whiskers) of two independent experiments (points) are shown. P value (Two-sided Mann-Whitney test) is indicated (***P < 0.001, n.s., not significant.). n = 20,428 transcripts. The boxplot indicates the median (middle line) and the third and first quartiles (box); the whiskers show the 1.5× IQR above and below the box. (c,d) Transcriptional response of the cells treated with THZ1/DMSO for 2h followed by 4sU labelling over 60 min. (c) MA plots comparing total gene expression level with log change in transcription per gene measured by 4-thioU RNA-seq (SLAM-seq). (d) MA plots comparing nascent gene expression levels with log change in transcription per gene measured by SLAM-seq. THZ1 treatment confirmed that transcripts containing T>C conversions of protein-coding genes were broadly repressed, which captured the prominent immediate responses, while the total mRNA level showed fewer changes. P-adjusted value by Wald test in DESeq2. (e) H3K4me3 levels on TSS before Auxin treatment of downregulated genes (n = 1,111) and unchanged genes (n = 10,107) measured by SLAM-seq (in response to Auxin treatment for 2h in DPY30–mAID cells). The p value was calculated with a two-sided Wilcoxon test. The boxplot indicates the median (middle line) and the third and first quartiles (box); the whiskers show the 1.5× IQR above and below the box. (f) H3K4me3 peak width of downregulated genes and unchanged genes measured by ChIP–seq at steady-state. n= for downregulated and n= for unchanged genes. The p value was calculated with a two-sided Wilcoxon test. The boxplot indicates the median (middle line) and the third and first quartiles (box); the whiskers show the 1.5× IQR above and below the box. (g) Box plot showing log2-transformed fold change of nascent transcription (SLAM-seq, 2 h vs. 0 h) of genes containing CGI (n = 11,386) and non-CGI (n = 3,262) promoters. The p value was calculated with a two-sided Wilcoxon test. The boxplot indicates the median (middle line) and the third and first quartiles (box); the whiskers show the 1.5× IQR above and below the box. (h) Gene set enrichment analysis (GSEA) of downregulated genes in DPY30–mAID cells in response to 2 h Auxin treatment.