Seq-Scope Protocol: Repurposing Illumina Sequencing Flow Cells for High-Resolution Spatial Transcriptomics.
Seq-Scope Protocol: Repurposing Illumina Sequencing Flow Cells for High-Resolution Spatial Transcriptomics.
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Seq-Scope 协议：重新利用 Illumina 测序流动槽实现高分辨率空间转录组学。
DOI：
10.1101/2024.03.29.587285
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发表时间：
2024
期刊：
影响因子：
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通讯作者：
Lee,JunHee
中科院分区：
文献类型：
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作者：
Kim,Yongsung;Cheng,Weiqiu;Cho,Chun-Seok;Hwang,Yongha;Si,Yichen;Park,Anna;Schrank,Mitchell;Hsu,Jer-En;Xi,Jingyue;Kim,Myungjin;Pedersen,Ellen;Koues,OliviaI;Wilson,Thomas;Jun,Goo;Kang,HyunMin;Lee,JunHee
空间转录组学 (ST) 技术代表了基因表达研究的重大进步，旨在从单个组织学切片中分析整个转录组。这些技术旨在克服免疫染色和 RNA 原位杂交等传统方法所面临的限制，这些方法只能同时分析少数靶基因。然而，ST在组织病理学分析中的应用也受到多种因素的限制，包括分辨率低、基因范围有限、可扩展性问题、成本高以及需要精密设备和复杂方法。 Seq-Scope——一项最近开发的新技术——重新利用 Illumina 测序平台进行高分辨率、高内容空间转录组分析，从而克服了这些限制。在这里，我们提供了使用 Illumina NovaSeq 6000 测序流动槽实施 Seq-Scope 的详细分步方案，可对 7 mm × 7 mm 或更大区域内的多个组织切片进行分析。除了详细说明如何准备用于组织学成像和测序文库制备的冷冻组织切片外，我们还提供全面的说明和简化的计算流程，以整合组织学和转录组数据以进行高分辨率空间分析。这包括使用传统的软件工具进行单细胞和空间分析，以及我们最近开发的用于以亚微米分辨率分析空间数据的无分割方法。鉴于其对各种生物组织的适应性，Seq-Scope 成为分子生物学和组织学研究人员的宝贵工具。
Spatial transcriptomics (ST) technologies represent a significant advance in gene expression studies, aiming to profile the entire transcriptome from a single histological slide. These techniques are designed to overcome the constraints faced by traditional methods such as immunostaining and RNA in situ hybridization, which are capable of analyzing only a few target genes simultaneously. However, the application of ST in histopathological analysis is also limited by several factors, including low resolution, a limited range of genes, scalability issues, high cost, and the need for sophisticated equipment and complex methodologies. Seq-Scope—a recently developed novel technology—repurposes the Illumina sequencing platform for high-resolution, high-content spatial transcriptome analysis, thereby overcoming these limitations. Here we provide a detailed step-by-step protocol to implement Seq-Scope with an Illumina NovaSeq 6000 sequencing flow cell that allows for the profiling of multiple tissue sections in an area of 7 mm × 7 mm or larger. In addition to detailing how to prepare a frozen tissue section for both histological imaging and sequencing library preparation, we provide comprehensive instructions and a streamlined computational pipeline to integrate histological and transcriptomic data for high-resolution spatial analysis. This includes the use of conventional software tools for single cell and spatial analysis, as well as our recently developed segmentation-free method for analyzing spatial data at submicrometer resolution. Given its adaptability across various biological tissues, Seq-Scope establishes itself as an invaluable tool for researchers in molecular biology and histology.