library_construction_protocol: All buffer compositions and reaction conditions followed the published protocol of Mahat and Kwak et al. (2016) unless otherwise specified. Briefly, 5 million K562 cells were mixed with 250,000 Drosophila S2 cells (5% spike-in), pelleted at 1000 r.c.f. for 5 min at 4°C, washed once with ice-cold PBS, resuspended with ice-cold permeabilization buffer at a density of 1 million cells/mL, and incubated on ice for 5 min. Cells were washed twice with the same volume of ice-cold permeabilization buffer, and resuspended in 100 μL storage buffer before immediate nuclear run-on or flash-freezing in liquid nitrogen for long-term storage at –80°C. Prior to the run-on reaction, 40 μL Dynabeads MyOne Streptavidin C1 beads (Thermo Fisher) per sample were pre-washed sequentially with Hydrolysis Buffer (0.1N NaOH + 50 mM NaCl), High Salt Wash Buffer, and Binding Buffer. Pre-washed beads were resuspended in 60 μL Binding Buffer per sample. The nuclear run-on reaction was performed at 37°C for 5 min with a final concentration of 20 µM each of Biotin-11-CTP, Biotin-11-UTP, ATP, and GTP. Following RNA extraction by Trizol LS (Invitrogen) and RNA fragmentation by base hydrolysis, 30 μL pre-washed C1 beads and 30 μL Binding Buffer were added to the ~60 μL RNA sample, and bead binding was performed at room temperature for 20 min on a rotational device. Beads were then washed twice using 500 μL High Salt Wash Buffer and once using 500 μL Low Salt Wash Buffer, with tube swap after each wash. Biotinylated RNA was eluted from beads by Trizol extraction, and 3' RNA adaptor ligation was performed in a total volume of 20 μL with 5 µM final adaptor concentration and 2 μL T4 RNA Ligase I, High Concentration (NEB). The reaction was incubated at 20°C for 4 h and held at 4°C overnight. On the next day, 50 μL Binding buffer and 30 μL pre-washed C1 beads were added to the reaction, and another round of bead binding and bead washing was performed as described above. Subsequent 5' enzymatic modifications of RNA were performed on beads with a reaction volume of 20 μL assuming 1 μL bead volume: 5' decapping reaction involved 1 μL RppH (NEB) and 1-h incubation at 37°C; 5' hydroxyl repair involved 1 μL T4 PNK (NEB) and 1-h incubation at 37°C. Beads were then washed once with 300 μL Binding Buffer, and 5' RNA adaptor ligation was performed on beads in a total volume of 20 μL with 5 µM final adaptor concentration and 2 μL T4 RNA Ligase I, High Concentration (NEB), incubated at room temperature for 1 h on a rotational device. Beads were washed twice using 500 μL High Salt Wash Buffer and once using 500 μL Low Salt Wash Buffer, with tube swap after each wash. RNA was eluted from beads by Trizol extraction and resuspended in 13 μL RT resuspension mix (8 μL DEPC H2O, 4 μL of 10 μM Illumina RP1 primer, 1 μL of 10 mM dNTPs). RNA was denatured at 65°C for 5 min and snap cooled on ice, and 7 μL RT master mix was added to the sample (4 μL of 5× RT Buffer, 1 μL of 100 mM DTT, 1 μL Invitrogen SUPERase·In RNase Inhibitor, 1 μL Thermo Scientific Maxima H Minus Reverse Transcriptase). RT reaction program was: 50°C for 30 min, 65°C for 15 min, 85°C for 5 min, hold at 4°C. The resulting cDNA was diluted with an equal volume of DEPC H2O. A test amplification was performed on 1:4 serial dilutions of 2 μL cDNA sample and run on 6% native PAGE TBE gel to determine the optimal PCR cycle number (N). Final amplification was performed in 100 μL volume (32.5 μL DEPC H2O, 20 μL of 5× Phusion HF Buffer, 20 μL of 5 M betaine, 2.5 μL of 10 µM Illumina RP1 primer, 2.5 μL of 10 µM Illumina indexing RPI-n primer, 2.5 μL of 10 mM dNTPs, 1 μL homemade Phusion polymerase, 19 μL cDNA sample). PCR program was: initial denaturation 95°C for 2 min; 5 cycles of 95°C for 30 s, 56°C for 30 s, 72°C for 30 s; N cycles of 95°C for 30 s, 65°C for 30 s, 72°C for 30 s; final extension 72°C for 5 min. PCR products were sequentially purified using the MinElute PCR purification kit (Qiagen) and homebrew SPRI beads (1.5× bead ratio) to remove all unused primers. DNA concentration was measured by the Qubit dsDNA High Sensitivity assay (Thermo Fisher). The libraries were pooled for sequencing on the Element Biosciences AVITI (2 × 80 bp paired-end sequencing) or Illumina NovaSeq 6000/X Plus platform (2 × 150 bp paired-end sequencing). Note that the 3' and 5' RNA adaptors contain a 6-nt unique molecular identifier (UMI) to enable accurate identification of PCR duplicates in downstream bioinformatic analysis. The complete sequences of the adaptors can be found in Judd et al., 2020.