Quantifying Transient RNA Dynamics

Newly synthesized RNA was pulse labeled by incubating confluent 10-cm culture dishes with 2 mM bromouridine (BrU; Sigma Aldrich) for 1 hour, either under normal culture conditions, under OND, or after 1 hour of recovery from OND. To estimate background incorporations, cells were analyzed without exposure to BrU. All experimental conditions were performed in triplicate. Following BrU pulse labeling, RNA was extracted using Trizol (Ambion) following the manufacturer’s instructions and following digestion to nucleosides with nuclease P1 (Roche), U snake venom phosphodiesterase (Worthington) and alkaline phosphatase (Fermentas) as described by Kellner et al. [77 (link)]. RNA nucleosides were subjected to liquid chromatography-mass spectrometry analysis. Separation was performed on an Agilent 1290 UHPLC system equipped with a ReproSil 100 C18 column (3 μm , 4.6 × 150 mm, Jasco GmbH) maintained at 30 °C. Identification and quantification of nucleosides was performed on an Agilent 6490 triple quadruple mass spectrometer.