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Rapid development in single-cell chromosome conformation capture technologies has provided valuable insights into the importance of spatial genome architecture for gene regulation. However, a long-standing technical gap remains in the simultaneous characterization of three-dimensional genomes and transcriptomes in the same cell. We have described an assay named Hi-C and RNA-seq employed simultaneously (HiRES), which integrates in situ reverse transcription and chromosome conformation capture (3C) for the parallel analysis of chromatin organization and gene expression. Here, we provide a detailed implementation of the assay, using mouse embryos and cerebral cortices as examples. The versatility of this method extends beyond these two samples, with the potential to be used in various other cell types.Key features• A multi-omics sequencing approach to profile 3D genome structure and gene expression simultaneously in single cells.• Compatible with animal tissues.• One-tube amplification of both DNA and RNA components.• Requires three days to complete.Graphical overviewSchematic illustration for the Hi-C and RNA-seq employed simultaneously (HiRES) workflow