Imaging the transcriptome in situ with high accuracy has been a major challenge in single cell biology, particularly hindered by the limits of optical resolution and the density of transcripts in single cells [1][2][3][4][5] . Here, we demonstrate seqFISH+, that can image the mRNAs for 10,000 genes in single cells with high accuracy and sub-diffraction-limit resolution, in the mouse brain cortex, subventricular zone, and the olfactory bulb, using a standard confocal microscope. The transcriptome level profiling of seqFISH+ allows unbiased identification of cell classes and their spatial organization in tissues. In addition, seqFISH+ reveals subcellular mRNA localization patterns in cells and ligand-receptor pairs across neighboring cells. This technology demonstrates the ability to generate spatial cell atlases and to perform discovery-driven studies of biological processes in situ. Spatial genomics, the analysis of the transcriptome and other genomic information directly in the native context of tissues, is crucial to many fields in biology, including neuroscience and developmental biology. Pioneering work in single molecule Fluorescence in situ Reprints and permissions information is available at www.nature.com/reprintsUsers may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://

Spatial transcriptomic and proteomic technologies have provided new opportunities to investigate cells in their native microenvironment. Here we present Giotto, a comprehensive and open-source toolbox for spatial data analysis and visualization. The analysis module provides end-to-end analysis by implementing a wide range of algorithms for characterizing tissue composition, spatial expression patterns, and cellular interactions. Furthermore, single-cell RNAseq data can be integrated for spatial cell-type enrichment analysis. The visualization module allows users to interactively visualize analysis outputs and imaging features. To demonstrate its general applicability, we apply Giotto to a wide range of datasets encompassing diverse technologies and platforms.

Fetal hemoglobin (HbF, αγ) level is genetically controlled and modifies severity of adult hemoglobin (HbA, αβ) disorders, sickle cell disease, and β-thalassemia. Common genetic variation affects expression of BCL11A, a regulator of HbF silencing. To uncover how BCL11A supports the developmental switch from γ- to β- globin, we use a functional assay and protein binding microarray to establish a requirement for a zinc-finger cluster in BCL11A in repression and identify a preferred DNA recognition sequence. This motif appears in embryonic and fetal-expressed globin promoters and is duplicated in γ-globin promoters. The more distal of the duplicated motifs is mutated in individuals with hereditary persistence of HbF. Using the CUT&RUN approach to map protein binding sites in erythroid cells, we demonstrate BCL11A occupancy preferentially at the distal motif, which can be disrupted by editing the promoter. Our findings reveal that direct γ-globin gene promoter repression by BCL11A underlies hemoglobin switching.

The rapid development of novel spatial transcriptomics technologies has provided new opportunities to investigate the interactions between cells and their native microenvironment. However, effective use of such technologies requires the development of innovative computational algorithms and pipelines. Here we present Giotto, a comprehensive, flexible, robust, and open-source pipeline for spatial transcriptomic data analysis and visualization. The data analysis module implements a wide range of algorithms ranging from basic tasks such as data pre-processing to innovative approaches for cell-cell interaction characterization. The data visualization module provides a user-friendly workspace that allows users to interactively visualize, explore and compare multiple layers of information. These two modules can be used iteratively for refined analysis and hypothesis development. We illustrate the functionalities of Giotto by using the recently published seqFISH+ dataset for mouse brain. Our analysis highlights the utility of Giotto for characterizing tissue spatial organization as well as for the interactive exploration of multi-layer information in spatial transcriptomic and imaging data. We find that single-cell resolution spatial information is essential for the investigation of ligandreceptor mediated cell-cell interactions. Giotto is generally applicable and can be easily integrated with external software packages for multi-omic data integration. Giotto facilitates the comprehensive analysis of single-cell spatial transcriptomic dataGiotto Analyzer is written in the popular language R. The core data structure is a simple and flexible S4 object ( Fig. 2A). Raw and processed count matrices are represented as a base matrix in R, while other annotations and metadata is encoded by an igraph network or a data.table. The former is a powerful library to work with networks, and the latter is a simple but intuitive table format with excellent performance for large-scale operations. In total, the Giotto uncovers different layers of spatial expression variabilityA key component of Giotto Analyzer is the implementation of a wide range of computational methods for spatial gene expression pattern identification. On a basic level, Giotto Analyzer can reduce the single-cell resolution data to a spatial grid through averaging (Supplementary Fig. 2A, B). Principal component analysis (PCA) is applied to the gridaverage data and significant principal components, along with their associated genes, are identified and reported. Using the aforementioned seqFISH+ dataset as an example, we found that the first principal component (PC) separates the outer layer extremities from the other layers. This is likely due to differences in cell-type compositions as most layers correlate with Slc17a7 expression, a marker for glutamatergic neurons, while the extremities show higher abundance of astrocytes and oligodendrocytes (Fig. 3A, top, Fig. 2D). In contrast, the second PC separates the outer and inner layers, which have similar cell-type composit...