The organization of mammalian genomes features a complex, multiscale three-dimensional (3D) architecture, whose functional significance remains elusive because of limited single-cell technologies that can concurrently profile genome organization and transcriptional activities. Here, we introduce genome architecture and gene expression by sequencing (GAGE-seq), a scalable, robust single-cell co-assay measuring 3D genome structure and transcriptome simultaneously within the same cell. Applied to mouse brain cortex and human bone marrow CD34⁺ cells, GAGE-seq characterized the intricate relationships between 3D genome and gene expression, showing that multiscale 3D genome features inform cell-type-specific gene expression and link regulatory elements to target genes. Integration with spatial transcriptomic data revealed in situ 3D genome variations in mouse cortex. Observations in human hematopoiesis unveiled discordant changes between 3D genome organization and gene expression, underscoring a complex, temporal interplay at the single-cell level. GAGE-seq provides a powerful, cost-effective approach for exploring genome structure and gene expression relationships at the single-cell level across diverse biological contexts.

哺乳动物基因组的组织具有复杂的多尺度三维（3D）架构，由于能够同时分析基因组组织和转录活动的单细胞技术有限，其功能意义仍然难以捉摸。在这里，我们介绍了基因组架构和基因表达测序 (GAGE-seq)，这是一种可扩展、稳健的单细胞联合测定，可在同一细胞内同时测量 3D 基因组结构和转录组。 GAGE-seq应用于小鼠大脑皮层和人骨髓 CD34 ⁺细胞，表征了 3D 基因组和基因表达之间的复杂关系，表明多尺度 3D 基因组特征告知细胞类型特异性基因表达并将调控元件与目标基因联系起来。与空间转录组数据的整合揭示了小鼠皮层的原位 3D 基因组变异。对人类造血作用的观察揭示了 3D 基因组组织和基因表达之间的不一致变化，强调了单细胞水平上复杂的、时间上的相互作用。 GAGE-seq 提供了一种强大且经济高效的方法，用于在不同生物背景下的单细胞水平上探索基因组结构和基因表达关系。