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.
Identifying the relationships between chromosome structures, chromatin states, and gene expression is an overarching goal of nuclear organization studies. Because individual cells are highly variable at all three levels, it is essential to map all three modalities in the same single cell, a task that has been difficult to accomplish with existing tools. Here, we report the direct super-resolution imaging of over 3,660 chromosomal loci in single mouse embryonic stem cells (mESCs) by DNA seqFISH+, along with 17 chromatin marks by sequential immunofluorescence (IF) and the expression profile of 70 RNAs, in the same cells. We discovered that the nucleus is separated into zones defined by distinct combinatorial chromatin marks. DNA loci and nascent transcripts are enriched at the interfaces between specific nuclear zones, and the level of gene expression correlates with an association between active or nuclear speckle zones. Our analysis also uncovered several distinct mESCs subpopulations with characteristic combinatorial chromatin states that extend beyond known transcriptional states, suggesting that the metastable states of mESCs are more complex than previously appreciated. Using clonal analysis, we show that the global levels of some chromatin marks, such as H3K27me3 and macroH2A1 (mH2A1), are heritable over at least 3-4 generations, whereas other marks fluctuate on a faster time scale. The longlived chromatin states may represent "hidden variables" that explain the observed functional heterogeneity in differentiation decisions in single mESCs. Our integrated spatial genomics approach can be used to further explore the existence and biological relevance of molecular heterogeneity within cell populations in diverse biological systems. MainCurrently, the main approaches to examine nuclear organization are 1) sequencing-based genomics, which measures contacts between DNA loci [1][2][3][4] and between DNA and nuclear bodies 5-10 , and 2) microscopy-based imaging of chromosomes in single cells, conventionally by multicolor DNA fluorescence in situ hybridization (DNA FISH) 11,12 . Genomics approaches have been powerful in mapping global contacts between chromosomes and have been scaled down to the single cell level [13][14][15][16][17][18][19] . However, reconstructing .
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...
Single molecule FISH (smFISH) has been the gold standard in quantifying individual transcripts abundances. Here, we demonstrate the scaling up of smFISH to the transcriptome level by profiling of 10,212 different mRNAs from mouse fibroblast and embryonic stem cells. This methods, called RNA SPOTs (Sequential Probing of Targets), provides an accurate and low-cost alternative to sequencing in profiling transcriptomes.RNA sequencing (RNAseq) 1,2 has been a powerful method to quantify RNAs in a diverse range of biological samples. While RNAseq has replaced microarrays as the de-rigueur method for genomics studies because of higher sensitivities and dynamic range, reverse transcription and other steps needed to convert RNA to cDNA to sequencing libraries can introduce biases in the quantitation of mRNAs. Moreover, sequencing the RNAs at nucleotide level is not necessary for counting the abundances of transcripts. Single molecule fluorescence in situ hybridization (smFISH) 3,4 , which directly hybridize DNA oligonucleotide probes to transcripts in cells, is highly sensitive and accurate in quantitating mRNA abundances. Here, we demonstrate transcriptome level profiling of mRNAs with single molecule sensitivity and high accuracy using a method based on sequential FISH (seqFISH) 5 . We had shown that seqFISH can be applied to image hundreds of transcripts in cells and tissues 6 , image dynamics of chromosomes 7 and allow lineage tracking with single cell resolution 8 . However, the major limitation of seqFISH is that optical diffraction limit prevents many mRNAs from being resolved simultaneously in single cells. In principle, super-resolution microscopy 9 and expansion microscopy 10 can resolve the optical density issue in situ.
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