{"id":244,"date":"2026-03-11T17:23:06","date_gmt":"2026-03-11T17:23:06","guid":{"rendered":"https:\/\/scienceweb.clemson.edu\/sparc\/?page_id=244"},"modified":"2026-03-13T18:40:13","modified_gmt":"2026-03-13T18:40:13","slug":"workflow","status":"publish","type":"page","link":"https:\/\/scienceweb.clemson.edu\/sparc\/workflow\/","title":{"rendered":"Workflow"},"content":{"rendered":"\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-a87de9ca alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<h3><strong>Stereo-seq Spatial Transcriptomics Workflow<\/strong><\/h3>\n<h4><span style=\"color: #000000\">The Stereo-seq workflow enables researchers to capture and analyze gene expression within tissues while preserving their spatial organization. This approach provides a powerful way to understand how cells interact and function within complex biological structures. The workflow consists of five main stages: sample preparation, staining and imaging, mRNA capture, library construction and sequencing, and computational analysis.<\/span><\/h4>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-b8998450\">\n<figure class=\"wp-block-image size-large is-resized\"><img fetchpriority=\"high\" decoding=\"async\" width=\"1024\" height=\"320\" src=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Workflow-1024x320.jpg\" alt=\"Workflow\" class=\"wp-image-290\" style=\"width:1180px;height:auto\" srcset=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Workflow-1024x320.jpg 1024w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Workflow-300x94.jpg 300w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Workflow-768x240.jpg 768w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Workflow-1536x480.jpg 1536w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Workflow.jpg 1656w\" sizes=\"(max-width: 1024px) 100vw, 1024px\" \/><\/figure>\n<\/div>\n<\/div><\/div>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-79960ed4 alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<div class=\"wp-block-uagb-container uagb-block-74324297\">\n<h3><strong> Sample Preparation<\/strong><\/h3>\n<h4><span style=\"color: #000000\">The workflow begins with the preparation of fresh frozen tissue sections, which are carefully placed onto a specialized Stereo-seq chip. This chip contains a high-density array of spatially barcoded capture probes that record the precise location of RNA molecules within the tissue. Proper tissue preservation during this stage is critical to maintain both RNA integrity and spatial structure.<\/span><\/h4>\n<h3><strong> Staining and Imaging<\/strong><\/h3>\n<h4><span style=\"color: #000000\">After mounting the tissue section, the sample is stained using ssDNA or H&amp;E staining to visualize the tissue morphology. High-resolution imaging is then performed using microscopy. These images serve as an anatomical reference that can later be aligned with spatial gene expression data, allowing researchers to directly link molecular information with tissue structure.<\/span><\/h4>\n<\/div>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-b293334f\">\n<figure class=\"wp-block-image size-large\"><img decoding=\"async\" width=\"1024\" height=\"761\" data-src=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/STaining-1024x761.jpg\" alt=\"STaining\" class=\"wp-image-303 lazyload\" data-srcset=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/STaining-1024x761.jpg 1024w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/STaining-300x223.jpg 300w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/STaining-768x571.jpg 768w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/STaining.jpg 1106w\" data-sizes=\"(max-width: 1024px) 100vw, 1024px\" src=\"data:image\/svg+xml;base64,PHN2ZyB3aWR0aD0iMSIgaGVpZ2h0PSIxIiB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciPjwvc3ZnPg==\" style=\"--smush-placeholder-width: 1024px; --smush-placeholder-aspect-ratio: 1024\/761;\" \/><\/figure>\n<\/div>\n<\/div><\/div>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-2714e22d alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<h3 data-start=\"0\" data-end=\"192\"><strong>Types and Sizes of Chips<\/strong><\/h3>\n<h4><strong data-start=\"0\" data-end=\"34\">P Chip (Permeabilization Chip): <\/strong><span style=\"color: #000000\">Used to optimize tissue <strong>permeabilization conditions<\/strong> prior to the spatial experiment, ensuring efficient RNA release while preserving tissue morphology.<\/span><\/h4>\n<h4 data-start=\"194\" data-end=\"406\"><strong data-start=\"194\" data-end=\"235\">T Chip (Spatial Transcriptomics Chip):\u00a0<\/strong><span style=\"color: #000000\">Used for <strong data-start=\"247\" data-end=\"279\" data-is-only-node=\"\">fresh-frozen tissue sections<\/strong>, where released mRNA binds to spatially barcoded probes on the chip, enabling high-resolution spatial gene expression mapping.<\/span><\/h4>\n<h4 data-start=\"408\" data-end=\"600\"><strong data-start=\"408\" data-end=\"452\">OMNI Chip (Spatial Transcriptomics Chip):\u00a0<\/strong><span style=\"color: #000000\">Designed for <strong data-start=\"468\" data-end=\"519\" data-is-only-node=\"\">FFPE (formalin-fixed paraffin-embedded) samples<\/strong>, enabling spatial transcriptomics from archived or clinically preserved tissues.<\/span><\/h4>\n<h4><span style=\"color: #000000\">\u2022 <strong>0.5 cm \u00d7 0.5 cm T Chip<\/strong>, which is typically used for smaller tissue sections or pilot spatial transcriptomics experiments.<\/span><\/h4>\n<h4><span style=\"color: #000000\"> \u2022 <strong>1 cm \u00d7 1 cm T Chip<\/strong>, which provides a larger capture area and enables profiling of larger tissue sections or multiple tissue regions simultaneously. <\/span><\/h4>\n<h4><span style=\"color: #000000\">\u2022 <strong>1 cm \u00d7 1 cm Omni Chip v1.1<\/strong>, which supports expanded spatial capture areas and improved experimental flexibility using FFPE samples.<\/span><\/h4>\n<h4><span style=\"color: #000000\"><strong>Ask questions for custom chips.<\/strong><\/span><\/h4>\n<\/div><\/div>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-67dadeea alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<figure class=\"wp-block-image size-large\"><img decoding=\"async\" width=\"1024\" height=\"238\" data-src=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Chip-types-1024x238.jpg\" alt=\"Chip types\" class=\"wp-image-310 lazyload\" data-srcset=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Chip-types-1024x238.jpg 1024w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Chip-types-300x70.jpg 300w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Chip-types-768x178.jpg 768w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Chip-types-1536x356.jpg 1536w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Chip-types-2048x475.jpg 2048w\" data-sizes=\"(max-width: 1024px) 100vw, 1024px\" src=\"data:image\/svg+xml;base64,PHN2ZyB3aWR0aD0iMSIgaGVpZ2h0PSIxIiB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciPjwvc3ZnPg==\" style=\"--smush-placeholder-width: 1024px; --smush-placeholder-aspect-ratio: 1024\/238;\" \/><figcaption class=\"wp-element-caption\">Chip types<\/figcaption><\/figure>\n<\/div><\/div>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-e161e04e alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<div class=\"wp-block-uagb-container uagb-block-4bb8363a\">\n<h3><strong>Permeabilization and In Situ mRNA Capture<\/strong><\/h3>\n<h4><span style=\"color: #000000\">Next, the tissue undergoes permeabilization, which allows messenger RNA (mRNA) molecules inside the cells to diffuse out and bind to the spatially barcoded probes on the chip surface. Each probe carries a unique positional barcode, ensuring that every captured transcript retains information about its original location within the tissue. This step effectively records the spatial distribution of gene expression across the sample.<\/span><\/h4>\n<\/div>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-bc6e30c7\">\n<figure class=\"wp-block-image size-full\"><img decoding=\"async\" width=\"1221\" height=\"621\" data-src=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Perma-3.jpg\" alt=\"Perma 3\" class=\"wp-image-306 lazyload\" data-srcset=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Perma-3.jpg 1221w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Perma-3-300x153.jpg 300w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Perma-3-1024x521.jpg 1024w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Perma-3-768x391.jpg 768w\" data-sizes=\"(max-width: 1221px) 100vw, 1221px\" src=\"data:image\/svg+xml;base64,PHN2ZyB3aWR0aD0iMSIgaGVpZ2h0PSIxIiB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciPjwvc3ZnPg==\" style=\"--smush-placeholder-width: 1221px; --smush-placeholder-aspect-ratio: 1221\/621;\" \/><\/figure>\n<\/div>\n<\/div><\/div>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-a3cef9d5 alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<h3><strong>Library Construction and Sequencing<\/strong><\/h3>\n<h4><span style=\"color: #000000\">Following RNA capture, the bound transcripts are converted into complementary DNA (cDNA) and prepared into sequencing libraries using the Stereo-seq visualization reagent set (PE75). These libraries are then sequenced using high-throughput sequencing platforms. During sequencing, both the gene identity and the spatial barcode information are read, enabling the reconstruction of spatial transcriptomic profiles.<\/span><\/h4>\n<\/div><\/div>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-116ad0f0 alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<figure class=\"wp-block-image aligncenter size-large\"><img decoding=\"async\" width=\"1024\" height=\"270\" data-src=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Lib-Prep-1024x270.jpg\" alt=\"Lib Prep\" class=\"wp-image-308 lazyload\" data-srcset=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Lib-Prep-1024x270.jpg 1024w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Lib-Prep-300x79.jpg 300w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Lib-Prep-768x203.jpg 768w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Lib-Prep.jpg 1517w\" data-sizes=\"(max-width: 1024px) 100vw, 1024px\" src=\"data:image\/svg+xml;base64,PHN2ZyB3aWR0aD0iMSIgaGVpZ2h0PSIxIiB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciPjwvc3ZnPg==\" style=\"--smush-placeholder-width: 1024px; --smush-placeholder-aspect-ratio: 1024\/270;\" \/><\/figure>\n<\/div><\/div>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-69b73b5c alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<h3><strong>Data Analysis and Visualization<\/strong><\/h3>\n<h4><span style=\"color: #000000\">Finally, the sequencing data are processed using computational pipelines such as SAW and StereoMap. These tools map sequencing reads back to their spatial coordinates and generate high-resolution maps of gene expression across the tissue. Researchers can then visualize spatial patterns of gene activity, identify cell types and functional regions, and study interactions between cells and tissues.<\/span><\/h4>\n<\/div><\/div>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<div class=\"wp-block-uagb-container uagb-block-086c337b alignfull uagb-is-root-container\"><div class=\"uagb-container-inner-blocks-wrap\">\n<figure class=\"wp-block-image aligncenter size-full is-resized\"><img decoding=\"async\" width=\"760\" height=\"365\" data-src=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Analysis-1.jpg\" alt=\"Analysis 1\" class=\"wp-image-296 lazyload\" style=\"--smush-placeholder-width: 760px; --smush-placeholder-aspect-ratio: 760\/365;width:974px;height:auto\" data-srcset=\"https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Analysis-1.jpg 760w, https:\/\/scienceweb.clemson.edu\/sparc\/wp-content\/uploads\/sites\/53\/2026\/03\/Analysis-1-300x144.jpg 300w\" data-sizes=\"(max-width: 760px) 100vw, 760px\" src=\"data:image\/svg+xml;base64,PHN2ZyB3aWR0aD0iMSIgaGVpZ2h0PSIxIiB4bWxucz0iaHR0cDovL3d3dy53My5vcmcvMjAwMC9zdmciPjwvc3ZnPg==\" \/><\/figure>\n<\/div><\/div>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Stereo-seq Spatial Transcriptomics Workflow The Stereo-seq workflow enables researchers to capture and analyze gene expression within tissues while preserving their 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center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-opacity":"","overlay-gradient":""}},"footnotes":""},"class_list":["post-244","page","type-page","status-publish","hentry"],"uagb_featured_image_src":{"full":false,"thumbnail":false,"medium":false,"medium_large":false,"large":false,"1536x1536":false,"2048x2048":false},"uagb_author_info":{"display_name":"mshahid","author_link":"https:\/\/scienceweb.clemson.edu\/sparc\/author\/mshahid\/"},"uagb_comment_info":0,"uagb_excerpt":"Stereo-seq Spatial Transcriptomics Workflow The Stereo-seq workflow enables researchers to capture and analyze gene expression within tissues while preserving their 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