(A) Drug condition analysis for actinomycin D and Cycloheximide in 293T cells. Transfected cells with tet-on GFP, were treated with different drugs and induced with Doxycycline at time 0 hr. Then cells were under time-course fluorescent detection. Cycloheximide (2 μg/ml) repressed translation, with low GFP level, Actinomycin D(5 μg/ml) blocked transcription, with no GFP expression detected. (B) Scatter plot for CSC scores from different human cell lines and/or methods measuring mRNA decay. (C) Scatter plots showing 293T endogenous CSC scores comparing to codon usage calculated from the genome, transcriptome (each gene with the weight of RPKM) or the top 500 highly expressed genes. Spearman correlation is used, p value and R value is indicated. (D) Boxplot showing the ratio of nucleotide transition without and with s4U treatment using the top five thousand expressed mRNA, only T_C transition gets significant increase after labeling, indicating the SLAM-seq labeling works. Wilcox rank sum test is used, p.value indicated. (E) Boxplot showing the T to C transition in each triplicate over time. The ratio decreased with time for the chasing period. (F) Scatter plot for mRNA-seq with regular RNA-seq and SLAM-seq in duplicates. Pearson correlation and P-values indicated. (G) Scatter plot for mRNA level with and without s4U treatment, s4U treatment did not influence gene expression. Pearson correlation and P-value indicated. (H) Scatter plot showing mRNA half-life of endogenous genes in K562 cell measured with two different methods, TimeLapse-seq (Schofield et al., 2018), and our SLAM-seq. Pearson correlation and p-value indicated. (I) Scatter plot showing CSC scores from our SLAM-seq data in K562 cells, published dataset for TimeLapse-seq in K562 (Schofield et al., 2018), and published SLAM-seq data in mouse embryonic stem cell (Herzog et al., 2017). Pearson correlation and p-value indicated.