Association between changes in PAL and mRNA abundance regardless of transcriptional changes. (A) CDF of ΔPAL (x-axis) with respect to transcriptional changes (ΔTXN; PRO-seq, log2 fold change) between 0 and 4 h post-activation. Transcripts are binned into six groups based on ΔTXN (e.g., (−4, −2] denoting −4 < ΔTXN ≤ −2), and their ΔPAL values are plotted as a group. K–S test P-value compares ΔPAL values from lowest and highest ΔTXN bins. TXN values (RPKM) were averaged from two biological replicates of PRO-seq at each time point, and the fold change value between two different time points was log2-transformed for ΔTXN calculation. (B) Box plots of ΔPAL for transcript isoforms with PAL changes out of the genes with minimal transcriptional changes (|ΔTXN| < 0.5 in all pairwise time intervals) for 0 and 4 h comparison. The number of transcripts is denoted. PAL changes were defined as |ΔPAL| ≥ 10 nt and K–S test FDR < 0.01. (C) Analysis of genes with minimal ΔTXN between 0 h through 4 h (|ΔTXN| < 0.5 in all pairwise time intervals). CDF of ΔPAL (x-axis) among the sets of transcripts grouped based on RNA abundance changes (ΔRNA; DN, NC, and UP) defined in Supplemental Figure S4F. K–S test P-value between DN and UP groups indicated. 3′-seq read count values were averaged from two biological replicates at each time point, and then the fold change between two different time points was log2-transformed for ΔRNA calculation. (D) CDF of ΔPAL (x-axis) differentiated among the three ΔRNA groups (ΔRNA; DN, NC, and UP). Transcripts grouped by ΔRNA proceeds with RSS by ΔTXN levels (between 0 and 4 h post-activation). Inset violin plot shows ΔTXN per ΔRNA group. K–S test P-value for comparison between DN and UP groups indicated. (E) CDF of ΔRNA/ΔTXN (x-axis; log2 [3′-seq FC/PRO-seq FC]) comparing 0 to 2 h (left) and 2 to 4 h (right), for genes grouped by ΔPAL between 0 and 2 h post-LPS (DN; ΔPAL < −10, NC; |ΔPAL| < 5, UP; ΔPAL > 10). K–S test P-values between ΔPAL UP and DN groups indicated. (F) ΔRNA/ΔTXN with respect to ΔPAL in transcriptionally up-regulated genes. ΔRNA/ΔTXN between 0 to 2 h (left) and 2 to 4 h (right), for genes grouped by ΔPAL (between 0 to 2 h as defined in E). Student's t-test P-values are denoted by asterisk. (G) ΔRNA/ΔTXN with respect to ΔPAL in transcriptionally down-regulated genes; otherwise as in F. (Significant differences indicated as follows: [*] P < 0.05; [**] P < 0.01; [***] P < 0.001). Number of transcript isoforms or genes for the corresponding bins or groups are in parentheses. (FC) Fold change, (RSS) random stratified sampling, (DN) down, (NC) no change, (UP) up. See also Supplemental Figures S4 and S5.
