library_construction_protocol: PRO-seq was performed based on the protocol provided by the Nascent Transcriptomics Core at Harvard Medical School, with some modifications. All procedures were carried out on ice, and all buffers were kept pre-chilled prior to sample preparation. In detail, HUDEP2 cell lines (clones 6F8 and 7C4), engineered to express the dTAG system, were seeded at a density of 0.5 × 106 cells/m in IMDM-based differentiation medium supplemented with cytokines and incubated for 24 hours. After differentiation, cells were treated with 100 nM dTAG-13 for 0.5, 3, or 6 hours. At each time point, cells were immediately harvested into 10 mL of ice-cold 1X DPBS and centrifuged at 1,500 rpm for 7 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 250 μL of Buffer W (10 mM Tris-HCl pH 8.0, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 10% glycerol, and 0.2 μL/mL SUPERase·In RNase inhibitor). Next, 10 mL of permeabilization Buffer P (Buffer W supplemented with 0.1% IGEPAL CA- 630, 0.05% Tween-20, and 0.2 μL/mL SUPERase-IN) was added carefully. Samples were incubated on ice for 5 minutes and centrifuged at 800 × g for 8 minutes at 4°C. The resulting permeabilized cells were washed with 10 mL Buffer W, centrifuged again under the same conditions, and resuspended in 400 μL of Buffer F (50 mM Tris-HCl pH 8.0, 40% glycerol, 5 mM MgCl2, 1.1 mM EDTA, 0.5 mM DTT, and 1 μL/mL SUPERase-IN RNase inhibitor; Thermo Fisher, Cat. No. AM26976). Cells were counted and QC at this step. Finally, cells were pelleted by centrifugation at 800 × g for 8 minutes at 4°C and resuspended in Buffer F at a concentration of 1 × 106 cells per 40 μL. Samples were flashfrozen immediately in liquid nitrogen (LN2) and stored at −80°C until further analysis. The Nascent Transcriptomics Core at Harvard Medical School, Boston, Massachusetts, conducted PRO-Seq library construction and data analysis, as previously reported  (Mimoso & Goldman, 2023).