Labeling and capture of nascent RNA

Click-iT Nascent RNA capture kit was used to detect newly synthesized RNA according to the manufacturer's instructions. 5-Ethymyl uridine (EU) is an alkyne-modified uridine analog and it is efficiently and naturally incorporated into nascent RNA. Cells were incubated in 0.2 mM of EU for 4 h and total RNA labeled with EU was isolated using Trizol reagent (Invitrogen). Then EU-labeled RNA was biotinylated in a Click-iT reaction buffer with 0.5 mM of biotin azide and subsequently captured on streptavidin magnetic beads.