Project description:Spatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology. Here we describe a simple method for transcriptional profiling of formalin fixed histology specimens based on mechanical isolation of tissue micro-regions containing 5-20 cells. Sequencing micro-regions from an archival melanoma specimen having multiple distinct histologies reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immunoediting and parallel imaging confirms changes in immuno-phenotypes and cancer cell states.
Project description:Spatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology. Here we describe a simple method for transcriptional profiling of formalin fixed histology specimens based on mechanical isolation of tissue micro-regions containing 5-20 cells. Sequencing micro-regions from an archival melanoma specimen having multiple distinct histologies reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immunoediting and parallel imaging confirms changes in immuno-phenotypes and cancer cell states.
Project description:Spatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology. Here we describe a simple method for transcriptional profiling of formalin fixed histology specimens based on mechanical isolation of tissue micro-regions containing 5-20 cells. Sequencing micro-regions from an archival melanoma specimen having multiple distinct histologies reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immunoediting and parallel imaging confirms changes in immuno-phenotypes and cancer cell states.
Project description:Expression profiling in spatially defined regions is crucial for systematically understanding tissue complexity. Here, we report a method of photo-irradiation for in-situ barcoding hybridization and ligation sequencing, named PBHL-seq, which allows targeted expression profiling from the photo-irradiated region of interest in intact fresh frozen and formalin fixation and paraffin embedding (FFPE) tissue samples. PBHL-seq uses photo-caged oligodeoxynucleotides for in situ reverse transcription followed by spatially targeted barcoding of cDNAs to create spatially indexed transcriptomes of photo-illuminated regions. We recover thousands of differentially enriched transcripts from different regions by applying PBHL-seq to OCT-embedded tissue (E14.5 mouse embryo and mouse brain) and FFPE mouse embryo (E15.5). We also apply PBHL-seq to the subcellular microstructures (cytoplasm and nucleus, respectively) and detect thousands of differential expression genes. Thus, PBHL-seq provides an accessible workflow for expression profiles from the region of interest in frozen and FFPE tissue at subcellular resolution with areas expandable to centimeter scale, while preserving the sample intact for downstream analysis to promote the development of transcriptomics.
Project description:Spatially resolved transcriptomics of tissue sections enables advances in fundamental and applied biomedical research. Here, we present Multiplexed Deterministic Barcoding in Tissue (xDBiT) to acquire spatially resolved transcriptomes of nine tissue sections in parallel. New microfluidic chips were developed to spatially encode mRNAs over a total tissue area of 1.17 cm2 with a 50 µm resolution. Optimization of the biochemical protocol increased read and gene counts per spot by one order of magnitude compared to previous reports. Furthermore, the introduction of alignment markers allowed seamless registration of images and spatial transcriptomic spots. Together with technological advances, we provide an open-source computational pipeline to prepare raw sequencing data for downstream analysis. The functionality of xDBiT was demonstrated by acquiring 16 spatially resolved transcriptomic datasets from five different murine organs, including the cerebellum, liver, kidney, spleen, and heart. Factor analysis and deconvolution of spatial transcriptomes allowed for in-depth characterization of the murine kidney.
Project description:The rapid development of spatial transcriptomics (ST) techniques has allowed the measurement of transcriptional levels across many genes together with the spatial positions of cells. This has led to an explosion of interest in computational methods and techniques for harnessing both spatial and transcriptional information in analysis of ST datasets. The wide diversity of approaches in aim, methodology and technology for ST provides great challenges in dissecting cellular functions in spatial contexts. Here, we synthesize and review the key problems in analysis of ST data and methods that are currently applied, while also expanding on open questions and areas of future development.
