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

Cell signaling plays a critical role in neurodevelopment, 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 cytoplasmic and nuclear proteins, including those with post-translational modifications, and to connect their activity with cis-regulatory elements and transcriptional targets. We utilize a simplified benchtop antibody conjugation method to create large custom neuro-focused antibody panels for simultaneous protein and scATAC-seq profiling on whole cells, alongside both experimental and computational strategies to incorporate transcriptomic measurements. We apply our workflow to cell lines, induced pluripotent stem cells, and months-old retinal and brain organoids to demonstrate its broad applicability. We show that Phospho-seq can provide insights into cellular states and trajectories, shed light on gene regulatory relationships, and help explore the causes and effects of diverse cell signaling in neurodevelopment. Phospho-seq Pilot: A mixed population of iPS cells and K562 Cells. The K562 cells consist of two populations, One treated with 50 ng/ml EGF for 15 mins and one treated with 500 nM PX-866 for four hours. The cells were harvested and prepared according to the Phospho-seq protocol and stained with ADTs and HTOs. Phospho-seq Benchmark: A mixed population of HEK and K562 cells were processed according to Phospho-seq protocol and stained with ADTs and HTOs Phospho-seq Benchmark_2: a mixed population of K562 cells treated with 50 ng/ml EGF and 500 nm PX-866 processed with the Phospho-seq protocol,stained with a pRPS6 ADT and a flourescent pRPS6 antibody and FACS sorted into quartile bins, where each population was satined with HTOs and run through the 10x Genomics scATAC-seq Protocol ASAP-seq Benchmark_2: a mixed population of K562 cells treated with 50 ng/ml EGF and 500 nm PX-866 processed with the ASAP-seq protocol,stained with a pRPS6 ADT and a flourescent pRPS6 antibody and FACS sorted into quartile bins, where each population was satined with HTOs and run through the 10x Genomics scATAC-seq Protocol Phospho-seq RetOrg: 34 week-old retinal organoids dissociated and processed with the Phospho-seq protocol, stained with ADTs and HTOs Phospho-multi: 18 and 24 week-old retinal organoids processed according to the Phospho-seq Multi protocol, stained with ADTs and constructed libraries using the 10x Genomics scATAC+scRNA kit Phospho-seq BrainOrg: 3-month old brain organoids dissociated and processed with the Phospho-seq protocol, stained with ADTs and HTOs Bridge-Multi: 3-month old brain organoids dissociated and processed with the according to the 10x Genomics scRNA and scATAC-seq kit Organoid RNA-seq reference: 3-month old brain organoids dissociated and stained with HTOs and processed with the 10x Genomics 3' v3.1 scRNA-seq processing protocol