Single‐Cell Annotation and Localization via Integrating Spatial Transcriptomics Maps the Mouse Ocular Atlas and RAO Dynamics

ABSTRACT Retinal artery occlusion (RAO), a blinding emergency, demands clarification of cellular spatiotemporal dynamics for targeted therapies. Single‐cell RNA‐seq is powerful but lacks spatial context, whereas spatial transcriptomics struggles with cell segmentation errors and low transcript capture efficiency. To overcome these, we developed the ASCAL (Automated Single‐Cell Annotation and Localization) pipeline, integrating complementary spatial transcriptomics techniques: single‐nucleus‐resolution SeekSpace for cell annotation reference and uniform‐coverage Stereo‐seq for localization of annotated cells. This strategy enables automated annotation and localization of large‐scale scRNA‐seq datasets with minimal spatial sections. Leveraging ASCAL, we constructed a mouse whole‐eye single‐cell spatial atlas precisely mapping cell types across the ciliary body and retina. Moreover, we revealed pronounced spatial immune activation specifically enriched in the ganglion cell layer (GCL) in the RAO model, further validated by immunofluorescence staining. We also uncovered a selective depletion of active Rods. Importantly, RNAscope assays independently validated both the peripheral localization of this active Rod subcluster and its pronounced loss following RAO. Overall, this study provides a comprehensive spatial cell atlas of the whole eye and offers novel spatial‐resolution insights into the pathological mechanisms of RAO, serving as a valuable resource for deciphering the physiological and pathological landscapes of the eye at single‐cell resolution.