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://

Eukaryotic genomes are packaged into a 3-dimensional structure in the nucleus. Current methods for studying genome-wide structure are based on proximity ligation. However, this approach can fail to detect known structures, such as interactions with nuclear bodies, because these DNA regions can be too far apart to directly ligate. Accordingly, our overall understanding of genome organization remains incomplete. Here, we develop split-pool recognition of interactions by tag extension (SPRITE), a method that enables genome-wide detection of higher-order interactions within the nucleus. Using SPRITE, we recapitulate known structures identified by proximity ligation and identify additional interactions occurring across larger distances, including two hubs of inter-chromosomal interactions that are arranged around the nucleolus and nuclear speckles. We show that a substantial fraction of the genome exhibits preferential organization relative to these nuclear bodies. Our results generate a global model whereby nuclear bodies act as inter-chromosomal hubs that shape the overall packaging of DNA in the nucleus.

Human adult spermatogenesis balances spermatogonial stem cell (SSC) self-renewal and differentiation, alongside complex germ cell-niche interactions, to ensure long-term fertility and faithful genome propagation. Here, we performed single-cell RNA sequencing of ~6500 testicular cells from young adults. We found five niche/somatic cell types (Leydig, myoid, Sertoli, endothelial, macrophage), and observed germline-niche interactions and key human-mouse differences. Spermatogenesis, including meiosis, was reconstructed computationally, revealing sequential coding, non-coding, and repeat-element transcriptional signatures. Interestingly, we identified five discrete transcriptional/developmental spermatogonial states, including a novel early SSC state, termed State 0. Epigenetic features and nascent transcription analyses suggested developmental plasticity within spermatogonial States. To understand the origin of State 0, we profiled testicular cells from infants, and identified distinct similarities between adult State 0 and infant SSCs. Overall, our datasets describe key transcriptional and epigenetic signatures of the normal adult human testis, and provide new insights into germ cell developmental transitions and plasticity.

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 .

Summary Visualization of the transcriptome and the nuclear organization in situ has been challenging for single cell analysis. Here, we demonstrate a multiplexed single molecule in situ method, intron seqFISH, that allows imaging of 10,421 genes at their nascent transcription active sites in single cells, followed by mRNA and lncRNA seqFISH and immunofluorescence. This nascent transcriptome profiling method can identify different cell types and states with mouse embryonic stem cells and fibroblasts. The nascent sites of RNA synthesis tend to be localized on the surfaces of chromosome territories and their organization in individual cells is highly variable. Surprisingly, the global nascent transcription oscillated asynchronously in individual cells with a period of 2 hours in mouse embryonic stem cells as well as in fibroblasts. Together, spatial genomics of the nascent transcriptome by intron seqFISH reveals nuclear organizational principles and fast dynamics in single cells that are otherwise obscured.