Cells were washed in PBS, then permeabilized and flash frozen in 1M cell aliquots. Permeabilized S2 cells were spiked with permeabilized HEK cells, then PRO-seq run-on reactions were performed using biotin-NTPs. Nascent RNA was extracted using a Total RNA Purification Kit following manufacturer instructions (Norgen Biotek Corp.). Extracted RNA was chemically fragmented with magnesium chloride and heat, followed by bead purication and trizol extraction of nascent RNA. Purified RNA was then subjected to 3' adaptor ligation, bead purification, 5' decapping, and 5' hydroxyl repair, followed by 5' adaptor ligation. Libraries were prepared by on-bead reverse transcription and 9-10 cycle amplification of final PRO-seq libraries. PRO-seq