48 h post transfection, media was aspirated from transfected plates and replaced with 10 mL of 500 nM 4-thio-uridine (4sU) containing ESC media and the plates incubated at 37°C with 5% CO2 for 5 min. After 5 min, the 4sU-containing media was aspirated and the cells were washed with PBS, trypsinized, and then pelleted by centrifugation. Total RNA was collected from cell pellets with a TRIzol extraction followed by an isopropanol/salt precipitation and resuspended in 100 µL 1XTE, according to ThermoFisher's recommendations. 100 µg of total RNA was diluted to a concentration of 240 ng/µL at a volume of 416.67 µL in 1XTE and then fragmented with a Bioruptor Pico (Diagenode) on high power for one 30 second cycle. The fragmented RNA was then separated using thiol-specific biotinylation followed by streptavadin bead treated to isolate 4sU-labelled RNA from total RNA. 1 ug of total RNA and 100 ng nascent RNA from each sample were used to build RNA-seq and TT-seq libraries, respectively. Total RNA was rRNA depleted using antisense oligos and purified. Total RNA-seq libraries were built using a strand-specific RNA-seq library build protocol.