Analysis of division-dependent global 3′-UTR changes in murine CD4+ T cells. (A) Schematic workflow of T cell-labeling, activation, and sorting. (B) Representative CTV-labeling profile of CD4+ T cells after in vitro activation for 48 h and typical results for sorted peaks of undivided T cells (peak 0), and T cells which are divided once (peak 1) and twice (peak 2). The right panel shows an overlay of all populations after FACS sorting. (C) Barplot representing the distribution of APA sites per gene. Pie plot shows the genomic localization distribution of APA sites. (D) PCA resulting from read coverage over the final peak set. (E) Stacked barplot summarizing 3′ UTRs identified by A-seq2 only (red), PacBio only (blue), or both A-seq2 and PacBio (green). (F) Example of Psen1 (chr12:83,732,374–83,735,700, “+”) gene 3′-UTR region. Overlap of A-seq2 peaks (consensus) and PolyASite database is labeled as the “Final peaks” track. A-seq2 signal of the different sorted cell populations is represented. Yellow boxes represent isoforms identified using PacBio long-read sequencing (arrows indicate newly identified isoforms), blue boxes represent Gencode (v25) isoforms, and black boxes indicate PAS.
