Project description:Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.
Project description:Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.
Project description:10X Genomics Xenium data from human Medulloblastoma samples. The data was acuired in the course of a study performing a comparison of four imaging-based ST methods – RNAscope HiPlex, Molecular Cartography, MERFISH/Merscope, and Xenium – together with sequencing-based ST (Visium). These technologies were used to study cryosections of medulloblastoma with extensive nodularity (MBEN), a tumor chosen for its distinct microanatomical features. Our analysis reveals that automated imaging-based ST methods are well suited to delineating the intricate MBEN microanatomy, capturing cell-type-specific transcriptome profiles. We devise approaches to compare the sensitivity and specificity of the different methods together with their unique attributes to guide method selection based on the research aim. Furthermore, we demonstrate how reimaging of slides after the ST analysis can markedly improve cell segmentation accuracy and integrate additional transcript and protein readouts to expand the analytical possibilities and depth of insights. This study highlights key distinctions between various ST technologies and provides a set of parameters for evaluating their performance. Our findings aid in the informed choice of ST methods and delineate approaches for enhancing the resolution and breadth of spatial transcriptomic analyses, thereby contributing to advancing ST applications in solid tumor research.
Project description:Spatial transcriptomics (ST) is fundamental for understanding molecular mechanisms in health and disease. Here, we present a protocol for efficient and high-resolution ST in 2D/3D with Open-ST. We describe all steps for repurposing Illumina flow cells into spatially barcoded capture areas and preparing ST libraries from stained cryosections. We detail the computational workflow for generating 2D/3D molecular maps ("virtual tissue blocks"), aligned with histological data, unlocking molecular pathways in space. Open-ST is applicable to any tissue, including clinical samples. For complete details on the use and execution of this protocol, please refer to Schott et al.1.
Project description:To investigate the effecs of commensal papillomavirus immunity on the homeostasis of highly mutated normal skin, spatial transcriptomics (Xenium, 10x Genomics, Pleasanton, CA) was performed on SKH-1 mouse back skin. The mice were treated with mouse papillomavirus (MmuPV1) or virus-like particles (VLP), followed by UV exposure for 25 weeks.
Project description:Xenium platform was used for the spatial transcriptomic analysis of human DRG neurons, 100 marker genes were selected as the customized probe panel and hybridized to fresh frozen hDRG sections. Manual segmentation of each neuron soma was performed, based on expressions of pan-neuronal marker gene PGP9.5, satellite glia cell marker FAB7B, and the corresponding H.E. staining. In total, 1340 neurons were identified (excluding 75 region-of-interest with poor or unclear neuronal soma morphology in H & E staining) and clustered into 16 groups. The 16 clusters were assigned as different cell types based on marker genes expression.
Project description:Spatial transcriptomics workflows using barcoded capture arrays are commonly used for resolving gene expression in tissues. However, existing techniques are either limited by capture array density or are cost prohibitive for large scale atlasing. We present Nova-ST, a dense nano-patterned spatial transcriptomics technique derived from randomly barcoded Illumina sequencing flow cells. Nova-ST enables customized, low cost, flexible, and high-resolution spatial profiling of large tissue sections. Benchmarking on mouse brain sections demonstrates significantly higher sensitivity compared to existing methods, at reduced cost.
Project description:Spatial transcriptomics (ST) technologies enable the mapping of gene expression to specific regions within tissues. However, current ST platforms present inherent trade-offs between resolution and throughput, which cannot be entirely addressed by a single technology. To unravel the spatiotemporal landscape of gene expression and cellular interactions during kidney injury and repair, we employed an integrated approach combining Xenium's high-resolution in situ sequencing with Visium's whole-transcriptome spatial profiling.