Determination of PAL with isoform specificity. (A) Schematic of activation time-course and sequencing strategy. (B) Validation of macrophage activation using qRT-PCR. In each sample, expression values were normalized to GAPDH expression. For each gene, plotted is fold change of gene expression (post-stimulation/unstimulated). Data is representative of two biological replicates, each performed with four technical replicates. (C) Structure of TED-seq libraries (top) and an illustration of PAL visualization by TED-seq on genome browser track (bottom). (D) Genome browser tracks of TED-seq (5′ terminus of reads) for SPSB1, indicating PAS previously annotated and determined by 3′-seq. Mean PAL (nt) and CPM values displayed for each 3′-UTR isoform in TED-seq and 3′-seq tracks, respectively. Arrow marks on top of the genome browser track indicate PAL from reference point for each 3′-UTR isoform. (E) Cumulative distribution (CDF) of spike-in PALs (x-axis). Representative data from a single library. The results from all time points are shown in Supplemental Figure S1B. (F) Histogram of PAS counts per gene. (G) PAS counts grouped by distance to reference PAS (nearest annotated PAS), for 3′-UTR sites and those within 1 kb downstream (n = 30,141). (H) Genome browser tracks of TED-seq and 3′-seq on ANTXR1 3′ UTR. Mean PAL (nt) and read count (CPM) displayed, with TED-seq read distributions magnified (inset box), and relative position of 3′-seq peaks to reference PAS shown under the PAS track (minus indicates upstream). (I) Genome browser tracks of TED-seq and 3′-seq on CD83 3′ UTR. De novo PAS isoforms track (PAS) shows the positions of PAS and their distances from annotated PAS, as in H. Mean PAL (nt) and read count (CPM) displayed as in C. Two biological replicates of TED-seq and 3′-seq libraries were prepared at each time point. One of two 0 h biological replicates is shown for TED-seq and 3′-seq data as a representative on the genome browser for D, H, and I. See also Supplemental Figure S1.
