STORM images were reconstructed using the ThunderSTORM plugin for FIJI (49). Maps of chromatin packing scaling (D) from raw PWS images were created using a custom analysis script in MATLAB that has been described in detail in previous works (21). Colocalization between PWS and STORM images was achieved through alignment of widefield reflectance images collected on each separate imaging arms. Errors caused by sample drift during imaging sequences were first corrected in ThunderSTORM; any additional corrections were applied manually as needed. To create plots in Fig. 4 and fig. S1, the average chromatin packing (D) was calculated from the PWS data (in red), and the average local RNAP II concentration was calculated from STORM data (in green) in each pixel (130 nm × 130 nm). Elongating RNAP II was labeled using the phospho-Ser2 antibody (ab193468). Data points with similar D are grouped together (D within 0.025) and plotted in Fig. 4D. The circles represent the means, and error bars are SE between regions.

To perform spatial analysis of PDs and Pol II locations, maps of D measured by PWS microscopy were first binarized using the function “imbinarize” in MATLAB with adaptive thresholding. This allows the segmentation of PDs. Next, the Euclidean distance transform of the binarized image was calculated using “bwdist” to find the distance between each pixel and the nearest PD. Positive distances denote pixels outside of PDs, and negative distances denote pixels inside of PDs. Next, all pixels containing no RNAP II (as visualized by STORM) were removed. The distances from pixels with RNAP II density higher than the mean (i.e., the most RNAP II–rich regions) were plotted in a violin plot (Fig. 4E).

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