Fig. 1

Profiling of transcript heterogeneity during three synchronized cell-fate transitions. a Schematic overview of the master time-course used for this study. Diploid cells harboring both IME1 fused to the CUP promoter and NDT80 expressed from the GAL promoter together with Gal4 fused to the estrogen receptor (pCUP-IME1 and GAL4.ER pGAL-NDT80) (FW2795) were grown in rich medium (YPD) overnight. Saturated cultures were pelleted, washed, and resuspended (OD₆₀₀ = 2.5) in sporulation medium (SPO). Samples were collected at the indicated time points, spanning the pre-meiotic (pm) cell-state and three different cell-fate transitions. Then, 50 μM CuSO₄ was added 2 h after the cells were transferred to SPO to induce IME1 expression and drive cells to enter meiosis (transition 1). Subsequently, 1 μM β-estradiol was added 6 h after transfer to SPO to induce NDT80 expression, which in turn induced meiotic divisions and spore formation (transition 2). In parallel, at 6 h in SPO, cells were returned to the mitotic cell cycle (transition 3) by transferring cells to YPD. b Evidence for synchrony of cell-fate transition 1, 2, and 3. For transition 1 (T1), the kinetics of pre-meiotic DNA replication was determined by flow cytometry analysis of DNA content (left panel). Samples were taken at indicated time points and fixed, and DNA content was measured by propidium iodide staining. For transition 2 (T2), kinetics of meiotic divisions was determined. Samples were taken at the indicated time point and fixed in ethanol, nuclei were stained with DAPI, and DAPI masses were counted. Cells that harbored two, three, or four DAPI masses were classified as cells undergoing meiosis I or meiosis II (% meiosis). In total, 200 cells were counted at each time point. For transition 3, budding kinetics was determined by cell morphology (right panel) for 200 cells per time point. Results are representative of three independent, biological repeats. c Schematic of sample collections, TSS-seq and TES-seq methods and other methods were used. In short, we performed mRNA-seq after total RNA extraction. In addition, poly(A) + RNA was purified from aliquots of the same total RNA, was fragmented, and was used as inputs for TSS-seq or TES-seq. For TSS seq, the fragments were dephosphorylated and treated with a decapping enzyme so that only bona fide mRNA 5′ ends were competent for ligation. A custom oligo was ligated to these ends and fragments were converted to cDNA libraries for sequencing. For TES-seq, fragments harboring the 3′ ends were converted to cDNA using a biotinylated, anchored oligo d(T) primer with a GsuI restriction enzyme site. cDNA was then captured on streptavidin beads and the poly(A) tails were shortened by GsuI, before library amplification and sequencing. For TIF-seq, equal amounts of total RNA from each time point spanning the pre-meiotic stage (pm) and each cell-fate transition (T1–3) were pooled. For MNase-seq, cells at selected time points were harvested to profile chromatin structure. The data represented are from n = 3 biological repeats. d Distribution of the numbers of unique TSSs/TESs at single nucleotide resolution per gene. e Overview of mRNA-seq (gray), TSS seq (red), and TES seq (blue) data at the RAD16 locus of different time points representing the different transitions (T1, T2, and T3). The scale of mRNA-seq, TSS-seq, and TES-seq values are depicted at the top of the panel. Scale (bp) are shown. f Distribution of the number of TSS/TES clusters per gene. g Percentage of TSS/TES clusters for each transition supported by TIF-seq. Weakly expressed TSSs/TESs (TPM < 10) are compared to the highly expressed ones (TPM ≥ 10). h Expression heatmap of genes known to be expressed early in gametogenesis (T1: RFA2, REC102, REC104, IME2), expressed after Ndt80 induction (T2: CLB3, CLB4, SPO12, SSP2) or expressed during mitotic growth (T3: RPL3, RPL27a, RPL32, RPL38). The pre-meiotic state (pm) is included as reference. mRNA-seq and TSS-seq and TES-seq data for each time point were scaled between 0 and 1 across the time course