### General information Author: Ana Faustino Mota, Szymon Berezicki, Erik Wernersson, Luuk Harbers, Xiaoze Li-Wang, Katarina Gradin, Christiane Peuckert, Nicola Crosetto, Magda Bienko Contact e-mail: magda.bienko@ki.se DOI: 10.17044/scilifelab.17080892 License: CC BY 4.0 This readme file was last updated: 11-10-2022 Please cite as: Faustino Mota, Ana; Wernersson, Erik; Li-Wang, Xiaoze; Gradin, Katarina; Crosetto, Nicola; Bienko, Magda (2021): FRET-FISH probes chromatin density at individual genomic loci in single cells (FRET-FISH datasets). SciLifeLab. Figure. 10.17044/scilifelab.17080892 ### Dataset description In eukaryotic cells, the density of DNA in the nucleus is thought to be intimately related to the regulation of gene expression. DNA accessibility is often used as a proxy for DNA density. However, it is not clear how DNA density and accessibility relate to each other, given the lack of tools for directly probing DNA density at defined genomic loci. To fill in this gap, here we developed FRET-FISH, a microscopy-based method combining fluorescence resonance energy transference (FRET) with DNA fluorescence in situ hybridization (FISH) to probe DNA density at defined genomic loci in single cells. We optimized FRET-FISH by testing different probe designs in situ and readily detecting FRET generated by DNA FISH probes in fixed cells. Additionally, we showed FRET FISH probes do not perturb the chromatin by measuring the distance between iFISH probes targeting flanking regions of the Ogt gene. To validate FRET-FISH, we tested whether it could detect differences in DNA density induced by various treatments, demonstrating its ability to detect local DNA density changes (i) upon treatment of cells with drugs that perturb chromatin condensation; (ii) during prolonged cell culture; and (iii) during the cell cycle (iv) along the nuclear radius. We compared FRET-FISH with ATAC-seq, demonstrating that the local DNA density and accessibility at defined loci are highly correlated (Pearson’s correlation coefficient: 0.88). We conclude that FRET-FISH is a sensitive tool for probing DNA density at defined genomic loci in single cells and for studying inter-allelic and cell-to-cell variability in chromatin density. ### Available variables A: Nuclei or cell identification number B: Field of view number C: Nuclei or cell identification number per field of view D: Number of signal pairs of donor and acceptor identified per nucleus E: Files location for donor F: Files location for acceptor G: Files location for FRET H: The x coordinate of the acceptor signal in pixel, multiply by 0.13 to get µm I: The y coordinate of the acceptor signal in pixel, multiply by 0.13 to get µm J: The z coordinate of the acceptor signal in pixel, multiply by 0.3 to get µm K: The x coordinate of the donor signal in pixel, multiply by 0.13 to get µm L: The y coordinate of the donor signal in pixel, multiply by 0.13 to get µm M: The z coordinate of the donor signal in pixel, multiply by 0.3 to get µm N: Intensity of the FRET signal O: Intensity of the Acceptor signal P: Intensity of the Donor signal Q: FRET pair identified per nuclei