Metabolic labeling of RNA for transcription rate determination

To assess relative RNA transcription rate, cells were treated with 1 mM 4-thiouridine (4sU; Sigma T4509) for exactly 10 min. Infection was stopped and RNA harvested using 1 ml TRIzol (Thermo Fisher), following manufacturer’s instructions. A fraction of the total RNA was reserved as input, and the remaining 4sU-labeled nascent RNA was biotinylated using MTSEA-Biotin-XX (Biotium, 90066) as previously described^67,81. Nascent RNA was separated from unlabeled RNA using MyOne C1 Streptavidin Dynabeads (Thermo Fisher Scientific, 65-001), biotin was removed from nascent RNA using 100 mM dithiothreitol (DTT), and RNA was isopropanol precipitated. One µg of total RNA (T) and an equivalent volume of nascent RNA (N) were converted to cDNA and qPCR was performed as described above. Relative transcription rates were determined by the ΔΔCt method to compare nascent transcript levels between control and siRNA treated cells normalized to nascent GAPDH RNA.