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Single\u2010cell RNA\u2010seq 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\u2010Cell Annotation and Localization) pipeline, integrating complementary spatial transcriptomics techniques: single\u2010nucleus\u2010resolution SeekSpace for cell annotation reference and uniform\u2010coverage Stereo\u2010seq for localization of annotated cells. This strategy enables automated annotation and localization of large\u2010scale scRNA\u2010seq datasets with minimal spatial sections. Leveraging ASCAL, we constructed a mouse whole\u2010eye single\u2010cell 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\u2010resolution insights into the pathological mechanisms of RAO, serving as a valuable resource for deciphering the physiological and pathological landscapes of the eye at single\u2010cell resolution.<\/jats:p>","DOI":"10.1002\/advs.75857","type":"journal-article","created":{"date-parts":[[2026,6,1]],"date-time":"2026-06-01T07:35:22Z","timestamp":1780299322000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Single\u2010Cell Annotation and Localization via Integrating Spatial Transcriptomics Maps the Mouse Ocular Atlas and RAO Dynamics"],"prefix":"10.1002","volume":"13","author":[{"given":"Chen","family":"Du","sequence":"first","affiliation":[{"name":"College of Life Sciences TaiKang Center for Life and Medical Sciences Department of Ophthalmology Renmin Hospital of Wuhan University Wuhan University  Wuhan 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