For experiments in (Supplementary Fig. 5B ), cells were transfected with 2.5 μg of the plasmid sfGFP-H2B-MRE-POLR3G-3'UTR or sfGFP-H2B-mut-POLR3G-3'UTR, as described in the “Transfection” section above. After 24 h, cells were fixed in 4% paraformaldehyde-PBS for 20 min at 37 °C then stained in 1:2000 diluted Hoechst 33232 (Thermo Fisher) for 10 min.
Image analysis was performed using custom code in Mathematica (Wolfram Research) to detect and measure the fluorescence intensities of all nuclei. Each background subtracted image in the Hoechst channel was binarized (using a sensible threshold) to create a nuclear mask. Using Mathematica’s built-in function “ComponentMeasurements,” nuclei that crossed the image edge were discarded, and masks were shape- and size-selected so that only whole, single nuclei were detected. The mask was applied to the 488 nm channel of the image and the total sfGFP fluorescence signal of each nucleus was measured. The imaging and analysis of all three experiments were performed blinded. All code is available on Github [https://github.com/Colorado-State-University-Stasevich-Lab/single-molecule-tracking-python ].
Image analysis was performed using custom code in Mathematica (Wolfram Research) to detect and measure the fluorescence intensities of all nuclei. Each background subtracted image in the Hoechst channel was binarized (using a sensible threshold) to create a nuclear mask. Using Mathematica’s built-in function “ComponentMeasurements,” nuclei that crossed the image edge were discarded, and masks were shape- and size-selected so that only whole, single nuclei were detected. The mask was applied to the 488 nm channel of the image and the total sfGFP fluorescence signal of each nucleus was measured. The imaging and analysis of all three experiments were performed blinded. All code is available on Github [