A cost-effective and flexible workflow for high-resolution spatial transcriptomics in fixed tissue
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ABSTRACT: The spatial heterogeneity of gene expression has driven the development of diverse spatial transcriptomics technologies. Here, we introduce photocleavage and ligation sequencing (PCL-seq), a spatial indexing method based on a light-controlled DNA labeling strategy applied to tissue sections. PCL-seq employs photocleavable oligonucleotides and sequence adapters to construct transcriptional profiles of specific regions of interest (ROIs), designated via microscopically controlled photo-illumination. In frozen mouse embryos, PCL-seq generates spatially aligned gene expression matrices and achieves high-quality data outputs, detecting approximately 170,000 unique molecular identifiers (UMIs) and 8,600 genes (irradiation diameter=100 µm). Moreover, PCL-seq is compatible with formalin-fixed and paraffin-embedded (FFPE) tissues, successfully identifying thousands of differentially enriched transcripts in the digits and vertebrae of FFPE mouse embryo sections. Additionally, PCL-seq achieves subcellular resolution, as demonstrated by differential expression profiling between nuclear and cytoplasmic compartments. These features establish PCL-seq as an accessible and versatile workflow for spatial transcriptomic analyses in both frozen and FFPE tissues at subcellular resolution.
Project description:We developed a method that utilizes floating mounting of thin sections of fixed frozen mouse lung tissue onto Xenium slides for the fluorescent in situ hybridization (FISH) and imaging–based spatial transcriptomics analysis of gene expression using the Xenium platform provided by 10X Genomics. Spatial transcriptomics techniques provide a comprehensive view by merging gene expression data with spatial context within their native tissue architecture. However, the Xenium pipeline has been validated only for formalin-fixed paraffin-embedded (FFPE) and fresh frozen sections by 10X Genomics. Notably, many researchers prefer paraformaldehyde-fixed cryosections for immunohistochemistry and in situ hybridization. In our study, we assessed the compatibility of standard fixed frozen mouse lung sections with the Xenium protocol. Our findings reveal that these sections not only align well with the Xenium platform but also offer superb imaging and gene expression quantification, even with limited number of genes in the Xenium panel. This protocol can serve as a valuable resource for preparing various tissues where FFPE and fresh frozen samples present challenges.
Project description:A total of four human melanoma liver metastasis (MLiM) formalin-fixed paraffin-embedded (FFPE) sections were analyzed using Nanostring GeoMx digital spatial profiling (NGDSP). The FFPE tissues were obtained from patients who underwent surgery and were pathological diagnosed with MLiM. Patients who had received any preoperative therapy were excluded. In each region of interest (ROI), two areas of interest (AOI)s were defined by HepPar1+ (hepatocytes); HMB45+ (melanoma cells) staining. A total of 43 AOIs were carefully reviewed for segments with sequencing saturation < 25%, nuclei fewer than 80, negative probe counts <3, or a surface area of <12,000 squared microns. Background correction and scaling were performed using geometric means and normalization was performed using Q3 averages of housekeeping genes. Genes with expression levels at or lower than limit of quantification (LOQ) in at least 5% of segments were filtered out. A total of 1,811 genes passed quality control and were analyzed. A total of seven ROIs with tumor margin (TM) and 13 ROIs with tumor center (TC) were selected, respectively. TM was defined as the areas on the border of malignant tumor cells close to normal tissue based on pathological features. TC was defined as central tumor tissue further inside from TM. In addition, 14 ROIs were selected at ANH. The ANH areas were defined as an area within 1,200 µm from the TM. Furthermore, nine ROIs were selected at the DNH areas. DNH was defined as an area at least 6 mm away from TM. Differential expressed genes (DEGs, Log2 fold-change >1.5, <-1.5, p < 0.05) were calculated on AOIs selected from MLiM comparing TC vs. TM; ANH vs. DNH; and ANH vs. Melanoma cells (TC+TM).
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 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.
Project description:Spatial transcriptomics facilitates the understanding of gene expression within complex tissue contexts. Among the array of spatial capture technologies available is 10x Genomics’ Visium which provides whole tissue section profiling, enabling whole transcriptome spatial analysis. Our dataset comprises spleen tissue from mice infected with malaria, spanning multiple experiments and sample preparation protocols for tissue preservation, either as fresh frozen at optimal cutting temperature (OCT) or formalin-fixed paraffin-embedded (FFPE). Tissue placement was also considered, comparing direct tissue placement on the slide with the use of CytAssist (CA), which expands the Visium platform’s capabilities by allowing for the pre-selection of tissue sections and genes through a set of probes. We also include a matching scRNA-seq dataset that can be integrated with the spatial data.
Project description:We performed spatial transcriptome profiling (ST-seq) on nine fresh frozen tissue sections to understand the immunophenotypes; immune cell types, states and their spatial location within the clear cell renal cell carcinoma (ccRCC) tumour microenvironment (TME).
Project description:Current spatial transcriptomic methods have been widely used to resolve gene expression; however, these methods are limited to fresh or fresh-frozen samples due to unsuccess of oligo(dT) primers in degraded RNA samples. Here we develop a spatial random-sequencing (spRandom-seq) technology for Formalin-fixed paraffin-embedded (FFPE) tissues by capturing full-length total RNAs with random primers. This approach provides a powerful spatial platform for clinical FFPE specimens and promises enormous applications in biomedicine.
Project description:Method to perform spatial analysis of mRNA in FFPE and PFA fixed tissue sections using spatially barcoded slides and based on oligo(dT) mRNA capture.
Project description:Formalin-fixed, paraffin-embedded (FFPE) tissues are banked in large repositories as a cost-effective means of preserving invaluable specimens for subsequent study, including for clinical proteomics in translational medicine. With the rapid growth of spatial proteomics, FFPE tissue samples can serve as a more accessible alternative to commonly used fresh frozen tissues. However, extracting proteins from FFPE tissue for analysis by mass spectrometry has been challenging due to crosslinks formed between proteins and formalin, particularly when studying limited samples. We have previously demonstrated that nanoPOTS (Nanodroplet Processing in One Pot for Trace Samples) is an enabling technology for high-resolution and in-depth spatial and single-cell proteomics measurements, but only fresh frozen tissues had been previously analyzed. Here we have adapted the nanoPOTS sample processing workflows for proteome profiling of 10-µm-thick FFPE tissues with lateral dimensions as small as 50 µm. Following a comparison of extraction solvents, times, and temperatures, and under the most favorable conditions, we respectively identified an average of 1180 and 2990 proteins from FFPE preserved mouse liver tissues having dimensions of 50 µm and 200 µm. This was on average 87% of the coverage achieved for fresh frozen mouse liver samples analyzed with the same general procedure. We also characterized the performance of our fully automated sample preparation and analysis workflow, termed autoPOTS, for FFPE spatial proteomics. These workflows provide the greatest depth of coverage reported to date for high-resolution spatial proteomics applied to FFPE tissues.
Project description:Mass spectrometry imaging dataset from fresh frozen mouse brain sections for development of a novel spatial segmentation computational pipeline.
Project description:Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays.This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin.