Co-profiling of in situ RNA-protein interactions and transcriptome in single cells and tissues [scMAPIT-seq]

RNA-binding proteins (RBPs) are essential regulators of RNA fate and function. A long-standing challenge in studying RBP regulation has been mapping RNA interactomes within the dynamic transcriptomic landscape, especially in single-cell contexts and primary tissues. Here we introduce MAPIT-seq (modification added to RBP interacting transcript-sequencing), which uses an antibody-directed editing strategy to map genome-wide in situ RBP–RNA interactions and gene expression concurrently. We demonstrate MAPIT-seq’s robustness across multiple RBPs and systematically analyze RNA substrates associated with core polycomb repressive complex 2 (PRC2) components. MAPIT-seq is also applicable to frozen tissue sections, enabling the mapping of RBP roles during brain development. Importantly, we develop high-throughput single-cell MAPIT-seq (scMAPIT-seq) to reveal cell stage-specific RBP regulation. In summary, MAPIT-seq expands multi-omics profiling, providing an effective framework to study post-transcriptional regulation in dynamic biological processes and clinically relevant scenarios. We advanced MAPIT-seq to a high-throughput single-cell format, uncovering cell cycle-specific G3BP1 targets and revealing heterogeneity in RNA-protein interactions at single-cell resolution. High-throughput single-cell MAPIT-seq was performed using DSP fixation in combination with the 10x Genomics platform.