FAIR sharing of Chromatin Tracing datasets using the newly developed 4DN FISH Omics Format

In recent years, multiplexed Fluorescence In Situ Hybridization (FISH) or FISH-omics methods have rapidly expanded, enabling the quantification of chromatin organization in single cells, often in conjunction with measurements of RNA and protein. These approaches have deepened our understanding of how 3D chromosome architecture relates to transcriptional activity and cell states in health and disease. Despite these advances, results from Chromatin Tracing FISH-omics experiments remain challenging to share, reuse, and analyze due to the absence of standardized data exchange specifications. Building on the release of microscopy metadata standards, we introduce the FISH Omics Format-Chromatin Tracing (FOF-CT), a community-developed standard for processed results from diverse imaging modalities. We describe the FOF-CT file format and present a curated collection of datasets deposited in the 4DN Data Portal and the OME Image Data Resource (IDR). We also highlight their potential for reuse, integration, and modeling by outlining example analysis pipelines and illustrating biological insights enabled by standardized, FAIR-compliant Chromatin Tracing datasets. While this manuscript focuses on the representation of ball-and-stick Chromatin Tracing, the format is designed to be extensible to volumetric Chromatin Tracing.