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. Overall design: 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

细胞信号转导在神经发育中发挥关键作用，可调控细胞行为与细胞命运。尽管多模态单细胞测序技术正快速发展，但同时对细胞信号转导状态及其他分子模态开展可扩展、灵活的谱型分析仍颇具挑战。本研究提出磷酸化测序（Phospho-seq）这一整合分析方法，旨在定量检测胞质与核蛋白（包括携带翻译后修饰的蛋白），并将其活性与顺式调控元件及转录靶标相关联。我们采用简化的台式抗体偶联方法，构建了定制化神经靶向抗体组，可在完整细胞中同时开展蛋白质与单细胞转座酶可及性测序（scATAC-seq）谱型分析，并结合实验与计算策略整合转录组测量数据。我们将该流程应用于细胞系、诱导多能干细胞（iPS细胞）以及培养数月的视网膜类器官与脑类器官，以证明其广泛适用性。研究结果表明，磷酸化测序可解析细胞状态与细胞轨迹，阐明基因调控关系，助力探索神经发育中多样细胞信号转导的因果机制。

实验整体设计：
Phospho-seq先导实验：诱导多能干细胞与K562细胞的混合群体。其中K562细胞分为两个亚群：一组经50 ng/ml表皮生长因子（EGF）处理15分钟，另一组经500 nM PX-866处理4小时。所有细胞均按照磷酸化测序流程进行收获与制备，并用抗体衍生标签（Antibody-Derived Tags, ADTs）与哈希标签寡核苷酸（Hash Tag Oligos, HTOs）染色。
Phospho-seq基准实验：HEK细胞与K562细胞的混合群体，按照磷酸化测序流程处理，并用ADTs与HTOs染色。
Phospho-seq基准实验2：经50 ng/ml EGF与500 nM PX-866处理的K562细胞混合群体，按照磷酸化测序流程处理，用pRPS6 ADT与荧光标记pRPS6抗体染色，并通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分为四分位群，每个亚群均用HTOs染色，随后按照10x Genomics scATAC-seq流程上机检测。
ASAP-seq基准实验2：经50 ng/ml EGF与500 nM PX-866处理的K562细胞混合群体，按照ASAP-seq流程处理，用pRPS6 ADT与荧光标记pRPS6抗体染色，并通过FACS分为四分位群，每个亚群均用HTOs染色，随后按照10x Genomics scATAC-seq流程上机检测。
Phospho-seq视网膜类器官实验：将34周龄的视网膜类器官解离后，按照磷酸化测序流程处理，并用ADTs与HTOs染色。
Phospho-multi实验：分别取18周与24周龄的视网膜类器官，按照磷酸化多模态测序流程处理，用ADTs染色，并使用10x Genomics scATAC+scRNA试剂盒构建文库。
Phospho-seq脑类器官实验：将3月龄的脑类器官解离后，按照磷酸化测序流程处理，并用ADTs与HTOs染色。
桥接多模态实验：将3月龄的脑类器官解离后，按照10x Genomics scRNA与scATAC-seq试剂盒流程进行处理。
类器官转录组测序参考数据集：将3月龄的脑类器官解离后，用HTOs染色，并按照10x Genomics 3' v3.1单细胞转录组测序流程进行处理。