The brain emerges from the primitive ectoderm as a sheet of neuroepithelial cells which folds into the neural tube during neurulation 1 . The developing nervous system is unique for the length of the developmental window, the extent of the interplay between different anatomical regions and lineages, and the diversity of cell types generated. Therefore, the ability of single-cell RNA-seq to disentangle the molecular heterogeneity of a complex cell pool has been particularly useful to study nervous system development [2][3][4][5][6][7][8][9][10] . Recent studies have shed light on the developing telencephalon 5,11 , the hippocampus 9,12,13 , the developing ventral midbrain 14-16, the developing spinal cord and cerebellum 17,18 , and the hypothalamic arcuate nucleus and diencephalon 19,20 . Single-cell RNA-seq has elucidated the differences between embryonic, postnatal and adult neural progenitors 9,21,22 , and compared normal glial progenitors with their malignant counterparts 23,24 .To map mouse brain development in detail, we collected embryonic brain tissue from 43 pregnant CD-1 mice, sampling each day from E7 to E18 (Extended Data Figure 1a-b, Table S1). We prepared 105 samples by droplet-based single-cell RNA sequencing. After removing low-quality cells and doublets (Methods), 96 samples remained with a mean of 5 766 transcripts (unique molecular identifiers, UMIs) and 1 934 genes detected per cell (Extended Data Figure 1c-f). The total cellular RNA content dropped as a function of

The human brain directs a wide range of complex behaviors ranging from fine motor skills to abstract intelligence and emotion. However, the diversity of cell types that support these skills has not been fully described. Here we used high-throughput single-nucleus RNA sequencing to systematically survey cells across the entire adult human brain in three postmortem donors. We sampled over three million nuclei from approximately 100 dissections across the forebrain, midbrain, and hindbrain. Our analysis identified 461 clusters and 3313 subclusters organized largely according to developmental origins. We found area-specific cortical neurons, as well as an unexpectedly high diversity of midbrain and hindbrain neurons. Astrocytes also exhibited regional diversity at multiple scales, comprising subtypes specific to the telencephalon and to more precise anatomical locations. Oligodendrocyte precursors comprised two distinct major types specific to the telencephalon and to the rest of the brain. Together, these findings demonstrate the unique cellular composition of the telencephalon with respect to all major brain cell types. As the first single-cell transcriptomic census of the entire human brain, we provide a resource for understanding the molecular diversity of the human brain in health and disease.

Methods to spatially profile the transcriptome are dominated by a trade-off between resolution and throughput. Here we develop a method named Enhanced ELectric Fluorescence in situ Hybridization (EEL FISH) that can rapidly process large tissue samples without compromising spatial resolution. By electrophoretically transferring RNA from a tissue section onto a capture surface, EEL speeds up data acquisition by reducing the amount of imaging needed, while ensuring that RNA molecules move straight down toward the surface, preserving single-cell resolution. We apply EEL on eight entire sagittal sections of the mouse brain and measure the expression patterns of up to 440 genes to reveal complex tissue organization. Moreover, EEL can be used to study challenging human samples by removing autofluorescent lipofuscin, enabling the spatial transcriptome of the human visual cortex to be visualized. We provide full hardware specifications, all protocols and complete software for instrument control, image processing, data analysis and visualization.

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Methods to spatially profile the transcriptome are dominated by a trade-off between resolution and throughput. Here, we developed a method named EEL FISH that can rapidly process large tissue samples without compromising spatial resolution. By electrophoretically transferring RNA from a tissue section onto a capture surface, EEL speeds up data acquisition by reducing the amount of imaging needed, while ensuring that RNA molecules move straight down towards the surface, preserving single-cell resolution. We applied EEL on eight entire sagittal sections of the mouse brain and measured the expression patterns of up to 440 genes to reveal complex tissue organisation. Moreover, EEL enabled the study of challenging human samples by removing autofluorescent lipofuscin, so that we could study the spatial transcriptome of the human visual cortex. We provide full hardware specification, all protocols and complete software for instrument control, image processing, data analysis and visualization.