Visualization of RNA Synthesis in Cells

Cells were incubated with 1 mM EU or 10 µM EdU (Berry & Associates, Inc.) for 30 min. Coverslips were fixed with 4% (w/v) PFA/PBS, and cells were permeabilized with 0.5% (v/v) Triton X-100/PBS. The click chemistry reaction was assembled with 10 mM sodium L-ascorbate (Sigma), 0.1 mM Biotin-TEG-azide (Berry & Associates, Inc.), and 2 mM Cu(II)SO4 (Sigma) in PBS. This was incubated at RT in the dark for 30 min. Following PBS washes, EU slides were incubated with primary antibodies against UBF and Nop52 as previously described (van Sluis and McStay 2015 (link)). Sites of synthesis were visualized with 1:200 Streptavidin conjugated TexasRed (Rockland) or Alexa647 (Jackson ImmunoResearch) for EU and EdU, respectively, combined with secondary antibodies (Jackson ImmunoResearch), diluted in 1% BSA/PBS for 1 h at 37°C.