Abstract Spatial transcriptomics aims to understand how the ensemble of RNA molecules in tissues and cells is organized in 3D space. Here we introduce Proximity RNA-seq, which enriches for nascent transcripts, and identifies contact preferences for individual RNAs in cell nuclei. Proximity RNA-seq is based on massive-throughput RNA-barcoding of sub-nuclear particles in water-in-oil emulsion droplets, followed by sequencing. We show a bipartite organization of the nuclear transcriptome in which compartments of different RNA density correlate with transcript families, tissue specificity and extent of alternative splicing. Integration of proximity measurements at the DNA and NA level identify transcriptionally active genomic regions with increased nucleic acid density and faster RNA polymerase II elongation located close to compact chromatin.

Spatial RNA proximities reveal a bipartite nuclear transcriptome and territories of differential density and transcription elongation rates