Figure 5. SM-PAT-seq decay analysis of total and ISG mRNAs.

Duplicate sets of LARP4 KO and WT MEFs were treated with IFNα, followed by transcription inhibition with actinomycin D, RNA isolation at multiple times thereafter, and analysis as follows. (A) Northern blot probed for ISG15 mRNA and 18S rRNA (top panel); stained gel prior to transfer (bottom panel). (B) Quantitation of biological duplicate northern data. ISG15 signal was normalized to 18S rRNA; t = 0 was set to 100%, error bars represent the spread. (C) Fraction of SM-PAT-seq CCS reads vs. PAT length for each time point after ActD addition for the gene set of ~10,500 mRNAs as in Figure 3A. (D) Fraction of SM-PAT-seq CCS reads vs. PAT length for each time point for the ISG set of 194 mRNAs. For C and D, the mitochondrial-encoded reads were filtered out.

Figure 5—figure supplement 1. Time course analysis of deadenylation by SM-PAT-seq.

(A) Box plots of median PAT lengths for total nuclear-encoded mRNAs in LARP4 KO and WT MEFs at various times after ActD as indicated above the tracks; boxes show 95% confidence intervals. (B) Box plots of median PAT lengths for ISG mRNAs in KO and WT as in A. (C) Box plots of median PAT lengths for the ISG mRNAs after sorting of reads based on PAT length as described in the text.