Simultaneous visualization of DNA loci in single cells by combinatorial multi-color iFISH

Single-molecule DNA fluorescence in situ hybridization (FISH) techniques have gained tremendous popularity as powerful tools to study the three-dimensional (3D) organization of the genome at the single cell level. However, there is an acute need for high quality, open access, curated and reproducible DNA FISH datasets. Here we describe a dataset obtained by applying our recently developed iFISH pipeline 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 colour coding can be used to precisely 3D localize multiple loci in single cells, and how inter-locus distances scale inversely with chromosome contact frequencies determined by Hi-C, as expected. We provide raw images and sub-pixel resolution 3D coordinates for nearly 10,000 dots. Our dataset provides a free resource that can be used to facilitate studies of 3D genome organization in single cells, gain deeper understanding of how FISH and Hi-C data can be integrated, and develop automatic FISH analysis algorithms.