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17Cell type identification is a key computational challenge in single-cell RNA-sequencing 18 (scRNA-seq) data. To capitalize on the large collections of well-annotated scRNA-seq datasets, 19 we present scClassify, a hierarchical classification framework based on ensemble learning. 20 scClassify can identify cells from published scRNA-seq datasets more accurately and more 21 finely than in the original publications. We also estimate the cell number needed for accurate 22 classification anywhere in a cell type hierarchy. 23 24Single cell RNA-sequencing (scRNA-seq) datasets have become larger and more diverse, driving the 25 need for classification methods that can provide more refined discrimination between cell types. 26Many major cell types can be divided into subtypes in a hierarchical fashion, forming what we call a 27 'cell type hierarchy' 1,2 . Approaches to scRNA-seq studies that account for cell type hierarchies in 28 experimental design and cell type identification will permit more nuanced interpretations of the 29 resulting data. 3 30 31The most common approach to identifying cell types within scRNA-seq data is unsupervised 32 clustering 4-6 followed by manual annotation using known marker genes. However, the number of 33

Advances in the study of neurological conditions have been possible due to induced pluripotent stem cell technologies and the generation of neural cell types and organoids. Numerous studies have described the generation of neural ectoderm-derived retinal and brain structures from pluripotent stem cells. However, the field is still troubled by technical challenges, including high culture costs and organoid-to-organoid variability. Here, we describe a simple and economical protocol that reproducibly gives rise to the neural retina and cortical brain regions from confluent cultures of stem cells. The spontaneously generated cortical organoids were isolated and cultured in suspension conditions for maturation and are transcriptionally comparable to organoids generated by other methods and to human foetal cortex. Furthermore, these organoids show spontaneous functional network activity with proteomic analysis and electron microscopy demonstrating the presence of synaptic components and maturity. The generation of retinal and brain organoids in close proximity also enabled their mutual isolation. Further culture of this complex organoid system demonstrated the formation of optic nerve-like structures connecting retinal and brain organoids, which might facilitate the investigation of the mechanisms of neurological diseases of the eye and brain.