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In this study, we extended co-indexing of transcriptomes and epitopes (CITE) to the spatial dimension and demonstrated high-plex protein and whole transcriptome co-mapping. We profiled 189 proteins and whole transcriptome in multiple mouse tissue types with spatial CITE sequencing and then further applied the method to measure 273 proteins and transcriptome in human tissues, revealing spatially distinct germinal center reactions in tonsil and early immune activation in skin at the Coronavirus Disease 2019 mRNA vaccine injection site.

We present spatial-CITE-seq for high-plex protein and whole transcriptome co-mapping, which was firstly demonstrated for profiling 198 proteins and transcriptome in multiple mouse tissue types. It was then applied to human tissues to measure 283 proteins and transcriptome that revealed spatially distinct germinal center reaction in tonsil and early immune activation in skin at the COVID-19 mRNA vaccine injection site. Spatial-CITE-seq may find a range of applications in biomedical research.

Spatial omics emerged as a new frontier of biological and biomedical research. Here, we present spatial-CUT&Tag for spatially resolved genome-wide profiling of histone modifications by combining in situ CUT&Tag chemistry, microfluidic deterministic barcoding, and next-generation sequencing. Spatially resolved chromatin states in mouse embryos revealed tissue-type-specific epigenetic regulations in concordance with ENCODE references and provide spatial information at tissue scale. Spatial-CUT&Tag revealed epigenetic control of the cortical layer development and spatial patterning of cell types determined by histone modification in mouse brain. Single-cell epigenomes can be derived in situ by identifying 20-micrometer pixels containing only one nucleus using immunofluorescence imaging. Spatial chromatin modification profiling in tissue may offer new opportunities to study epigenetic regulation, cell function, and fate decision in normal physiology and pathogenesis.

Summary This protocol describes the use of the deterministic barcoding in tissue for spatial omics sequencing platform to construct a multi-omics atlas on fixed frozen tissue samples. This approach uses a microfluidic-based method to introduce combinatorial DNA oligo barcodes directly to the cells in a tissue section fixed on a glass slide. This technique does not directly resolve single cells but can achieve a near-single-cell resolution for spatial transcriptomics and spatial analysis of a targeted panel of proteins. For complete details on the use and execution of this protocol, please refer to Liu et al. (2020)., Graphical abstract, Highlights • Simultaneous transcriptomic and proteomic analysis on fixed tissue slides • 10- and 25-μm spot size for varying resolution and near-single-cell analysis • DBiT-seq can be conducted with common laboratory equipment and reagents • DBiT-seq produces high-quality RNA sequencing data with high spatial resolution (10 μm), This protocol describes the use of the deterministic barcoding in tissue for spatial omics sequencing platform to construct a multi-omics atlas on fixed frozen tissue samples. This approach uses a microfluidic-based method to introduce combinatorial DNA oligo barcodes directly to the cells in a tissue section fixed on a glass slide. This technique does not directly resolve single cells but can achieve a near-single-cell resolution for spatial transcriptomics and spatial analysis of a targeted panel of proteins.

Spatial biology is emerging as a new frontier of biomedical research in development and disease, but currently limited to transcriptome and a panel of proteins. Here we present spatial epigenome profiling for three histone modifications (H3K27me3, H3K4me3, H3K27ac) via next-generation sequencing by combining in-tissue CUT&Tag chemistry and microfluidic deterministic barcoding. Spatial chromatin states in mouse embryos or olfactory bulbs revealed tissue type-specific epigenetic regulations, in concordance with ENCODE reference data, but providing spatially resolved genome-wide profiles at tissue scale. Using fluorescence imaging to identify the tissue pixels (20μm) each containing one nucleus allowed us to extract single-cell epigenomes in situ. Spatial chromatin state profiling in tissue may enable unprecedented opportunities to study epigenetic regulation, cell function and fate decision in normal physiology and pathogenesis.

SUMMARYWe present DBiT-seq –Deterministic Barcoding in Tissue for spatial omics sequencing – for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via NGS sequencing. Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide and crossflow of two sets of barcodes A1-50 and B1-50 followed by ligationin situyielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10μm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.In BriefMicrofluidic deterministic barcoding of mRNAs and proteins in tissue slides followed by high-throughput sequencing enables the construction of a high-spatial-resolution multi-omics atlas at the genome scale. Application to mouse embryos (E10-12) identified major tissue types in early organogenesis and revealed fine tissue features such as retinal pigmented epithelium and endothelial microvasculature at the cellular level.HighlightsDeterministic barcoding in tissue enables NGS-based spatial multi-omics mapping.DBiT-seq identified spatial patterning of major tissue types in mouse embryos.DBiT-seq revealed fine features such as retinal pigmented epithelium and microvascular endothelium at the cellular level.Direct integration with scRNA-seq data allows for rapid cell type identification.

We present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via next-generation sequencing (NGS). Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, A1-50 and B1-50, followed by ligation in situ, yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10-μm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.