Fig. 2

Alternative TSSs and TESs are pervasive expressed during T1, T2, and T3. a Schematic depicting the main and alternative TSSs (red) and TESs (blue) nearby a gene prior to and during cell-fate transitions (top). Definition of main and alternative TSS and TES (bottom). The main TSS/TES for T1 was defined as the most used TSS/TES in pre-meiotic cells (2 h). The main TSS/TES for T2 and 3 was defined as the most used TSS/TES during meiotic prophase (6 h). Any other TSS/TESs associated with the same gene were classed as alternative. TSS/TES clusters were only defined for a transition if they had a minimum level of expression (TPM ≥ 1) and were in the same orientation as the gene. b Number of distinct main (m) or alternative (a) TSS/TES clusters associated with genes for each cell-fate transition (T1, T2, and T3). c Distribution of 5′ (left) and 3′ (right) UTR lengths for main (m) or alternative (a) TSSs and TESs. The 5′ UTR length is defined as the distance given in number of nucleotides from the apex of a TSS cluster to the AUG of an annotated ORF. The 3′ UTR length is defined as the distance given in number of nucleotides from the apex of a TES cluster to the stop codon of an annotated ORF. Violin plots were scaled to a constant width. The alternative TSSs/TESs which were external to the ORF sequence and upregulated (two-fold or more) during T1 (n = 1118 for TSSs, and n = 1343 for TESs), T2 (n = 1079 for TSSs, and n = 1320 for TESs), or T3 (n = 1052 for TSSs, and n = 1611 for TESs) were used for this analysis. For the main TSSs/TESs n = 5016 and 5285 data points were used. The median position of main TSSs was 75 bp (T1) and 77 bp (T2 and T3), while that of the alternative TSSs upregulated during cell-fate transitions (2-fold or more) were at 170 bp (T1), 173 bp (T2) and 112 bp (T3) respectively. d Formulas for calculating alternative TSS/TES usage and alternative TSS/TES usage changes. Alternative TSS/TES usage for a gene was calculated by taking the alternative TSS/TES values divided over the sum of the main TSS/TES and alternative TSS/TES values. Alternative TSS/TES usage change was calculated by taking the difference in alternative TSS/TES usage between the transition (T) time point and the reference time point prior (PT) to transition. e Boxplots of alternative TSS and TES usage across different time points in T1, T2, and T3 using the formula defined in d. Negative controls (3 h, mock treated (3M) and 7 h, mock treated (7M)) representing cells which were shifted to SPO, but without inducing T1 or T2, were included. The alternative TSSs/TESs upregulated (two-fold or more) during T1, T2, or T3 were used for the analysis. f Similar to e, except that violin plots of changes in alternative TSS usage at different time points are displayed. Samples were compared using the Wilcoxon rank-sum test and * denotes p < 0.05