A Chromatin Fiber Analysis Pipeline to Model DNA Synthesis and Structures in Fission Yeast.
Chromatin fibers, first described by Jackson and Pombo (J Cell Biol 140(6):1285-1295, 1998) are prepared from cells lysed on glass coverslips, and require minimal equipment to produce. Since the DNA is not previously treated with denaturing agents, proteins are left intact and may be used to model other DNA-based processes. Such an analysis can be daunting, without a rigorous method for analysis. We describe a pipeline for chromatin fiber use to model DNA replication complexes. Full protocols for chromatin fiber preparation and staining are presented. Further, we have developed an analysis algorithm for One Dimensional Data-Boolean Logic Operations Binning System (ODD-BLOBS). This freely available software defines replication and protein tracts, measures their lengths, and then correlates replicated areas with protein distributions. Our methods and analysis are tested in Schizosaccharomyces pombe (fission yeast) but may be applied to model replication structures across multiple organisms.
Sabatinos SA, Green MD