### General information Author: Ana Faustino Mota, Maud Schweitzer, Magda Bienko Contact e-mail: magda.bienko@ki.se DOI: 10.17044/scilifelab.14267795 License: CC BY 4.0 This readme file was last updated: 11-06-2021 Please cite as: Faustino Mota, Ana; Schweitzer, Maud; Bienko, Magda (2021): Simultaneous visualization of DNA loci in single cells by combinatorial multi-color iFISH (Dataset 2: miFISH chr2 Replicate 2). SciLifeLab. Figure. https://doi.org/10.17044/scilifelab.14267795 ### Dataset description Single-molecule DNA fluorescence in situ hybridization (FISH) techniques enable studying the three-dimensional (3D) organization of the genome at the single cell level. However, there is a major unmet need for open access, high quality, curated and reproducible DNA FISH datasets. Here, we describe a dataset obtained by applying our recently developed iFISH method to simultaneously visualize 16 small (size range: 62–73 kilobases, kb) evenly spaced DNA loci on chromosome 2 in human cells, in a single round of hybridization. We show how combinatorial color coding can be used to precisely localize multiple loci in 3D within single cells, and how inter-locus distances scale inversely with chromosome contact frequencies determined by high-throughput chromosome conformation capture (Hi-C). We provide raw images and sub-pixel resolution 3D coordinates for nearly 10,000 FISH dots. Our dataset provides a free resource that can facilitate studies of 3D genome organization in single cells and can be used to develop automatic FISH analysis algorithms. ### Available variables A: Name of the file and field of view B: Channel name correspond to the equivalent colour C: Nuclei or cell identification number D: The x coordinate of the dot in pixel, multiply by 0.13 to get µm E: The y coordinate of the dot in pixel, multiply by 0.13 to get µm F: The z coordinate of the dot in pixel, multiply by 0.2 to get µm G: Intensity value of the pixel H: Full weight half maximum I: Signal-to-noise ratio J: Label of the dot that belongs to the allele 1 or 2 K: Dilation of the nucleus staining segmentation L: Lamina distance in pixels Lamina distance normalised M: Nucleus centre distance in pixels Nucleus centre distance normalised N: Normalised x position in the nucleus O: Normalised y position in the nucleus P: Normalised z position in the nucleus