Phospho-seq: Integrated, multi-modal profiling of intracellular protein dynamics in single cells.

Cell signaling plays a critical role in regulating cellular behavior and fate. While multimodal single-cell sequencing technologies are rapidly advancing, scalable and flexible profiling of cell signaling states alongside other molecular modalities remains challenging. Here we present Phospho-seq, an integrated approach that aims to quantify phosphorylated intracellular and intranuclear proteins, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, and integrate this information with scRNA-seq datasets via bridge integration. We apply our workflow to cell lines, induced pluripotent stem cells, and 3-month-old brain organoids to demonstrate its broad applicability. We demonstrate that Phospho-seq can define cellular states and trajectories, reconstruct gene regulatory relationships, and characterize the causes and consequences of heterogeneous cell signaling in neurodevelopment.