5-EU incorporation assay for RNA synthesis

Cells were plated on poly-D-Lys coated coverslip 24 h before labeling. At the time of the assay, cells were incubated with complete medium with 0.5 mM EU for 1 h. Cells were then washed twice with DPBS, fixed with 4% formaldehyde (v/v) in DPBS for 15 min and permeabilized with 0.5% Triton X-100 in DPBS for 15 min. After permeabilization, cells were washed once with 3%BSA in DPBS, and then incubated with click-reaction cocktail made with Click-&-Go Cell Reaction Buffer Kit and 2.5μM AFDye 488 Azide (Click Chemistry Tools) for 30 min. After reaction, cells were washed once with 3%BSA in DPBS and once with DPBS. Coverslips were mounted on slides with ProLong Gold Antifade Mountant with DAPI and imaged by ZEISS LSM 800. Random regions were imaged, and the quantification is as described in “Quantitative image analysis”.