Project description:Here we demonstrate brain epigenomic tomography that is constructed via epigenomic profiling of mouse neocortex slices with sub-millimeter thickness using a low-input ChIP-seq technology. We profiled the variation in histone modification signal (H3K27me3 and H3K27ac) across these slices and grouped the spatial epigenomic features into clusters of various spatial variation patterns.
Project description:Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using for example in situ hybridization, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially-resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly, our method enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs. To generate spatially-resolved RNA-seq data for zebrafish embryos (shield stage, 10 somites, 15 somites, 18 somites) and mouse forelimbs (E10.5), we cryosectioned samples, extracted RNA from the individual sections, and amplified and barcoded mRNA using the CEL-seq protocol (Hashimshony et al., Cell Reports, 2012) with a few modifications. Libraries were sequenced on Illumina HiSeq 2500 using 50bp paired end sequencing. Selected zebrafish libraries were sequenced on MiSeq 250bp paired-end to improve 3' annotations.
Project description:Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. While genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using for example in situ hybridization, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. We also demonstrate that our technique is suitable for spatially-resolved differential expression analysis in wildtype and Gli3 mutant mouse forelimbs. Importantly, our method enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.
Project description:During pneumonic plague, the bacterium Yersinia pestis elicits the development of inflammatory lung lesions that continue to expand throughout infection. This lesion development and persistence is poorly understood. Here, we examine spatially distinct regions of lung lesions using laser capture microdissection and RNAseq analysis to identify transcriptional differences between lesion microenvironments. We show that cellular pathways involved in leukocyte migration and apoptosis are down regulated in the center of lung lesions compared to the periphery. Probing for the bacterial factor(s) important for the alteration in neutrophil survival, we show both in vitro and in vivo that Y. pestis increases neutrophil survival in a manner that is dependent on the type-III secretion system effector YopM. This research explores the complexity of spatially distinct host - microbe interactions and emphasizes the importance of cell relevance in assays in order to fully understand Y. pestis virulence. We examine spatially distinct regions of lung lesions using laser capture microdissection and RNAseq analysis to identify transcriptional differences between lesion microenvironments. Sample types: uninfected BM-PMN, infected BM-PMN, lesion periphery, lesion center.
Project description:The recent development of spatial omics enables single-cell profiling of the transcriptome and the 3D organization of the genome in a spatially resolved manner. A spatial epigenomics method would expand the repertoire of spatial omics tools and accelerate our understanding of the spatial regulation of cellular processes and tissue functions. Here, we developed an imaging approach for spatially resolved profiling of epigenetic modifications in single cells