Fig. 3: Well-TEMP-seq characterizes the transcriptome dynamics of HCT116 cells in 5-AZA-CdR response.

a Experimental scheme of characterizing the transcriptome dynamics of 5-AZA-CdR-treated HCT116 cells with Well-TEMP-seq. Cells were treated with 300 nM 5-AZA-CdR for 0/1/2/3 d. Cells in each group were labeled with 200 μM 4sU for 2 h before fixation for Well-TEMP-seq. b Box plot showing the fraction of labeled transcripts per cell in different groups before correction. Here, correction is essential since current metabolic RNA labeling strategies may cause incomplete 4sU labeling of newly transcribed RNAs due to the presence of pre-existing uridine in cells and false positives arising from PCR errors and sequencing. c Box plot of detection rate of different groups. Calculated as the ratio between observed and corrected newly transcribed RNA levels for each cell, the detection rate is an important coefficient in the correction process and represents the labeling efficacy of newly transcribed RNAs. d Box plot showing the fraction of new transcripts per cell in different groups after correction. In b–d, n = 872 (0 d), 867 (1 d), 884 (2 d), 832 (3 d) cells and boxplots include centerline, median; box limits, upper and lower quartiles; and whiskers are highest and lowest values no >1.5× interquartile range. e Heat maps showing average new RNA levels (z-scaled natural log transformation of (TP10K + 1)) of significantly up-regulated genes (left) and down-regulated genes (right) in response to 5-AZA-CdR treatment. Source data are provided as a Source Data file.