Quantitative Analysis of PcG-Associated Condensates by Stochastic Optical Reconstruction Microscopy (STORM).

Abstract

The polycomb group (PcG) proteins play a central role in the maintenance of a repressive state of gene expression. Recent findings demonstrate that PcG components are organized into nuclear condensates, contributing to the reshaping of chromatin architecture in physiological and pathological conditions, thus affecting the nuclear mechanics. In this context, direct stochastic optical reconstruction microscopy (dSTORM) provides an effective tool to achieve a detailed characterization of PcG condensates by visualizing them at a nanometric level. Furthermore, by analyzing dSTORM datasets with cluster analysis algorithms, quantitative information can be yielded regarding protein numbers, grouping, and spatial organization. Here, we describe how to set up a dSTORM experiment and perform the data analysis to study PcG complexes' components in adhesion cells quantitatively.