AbstractSpatial transcriptomics technologies aim to advance gene expression studies by profiling the entire transcriptome with intact spatial information from a single histological slide. However, the application of spatial transcriptomics is limited by low resolution, limited transcript coverage, complex procedures, poor scalability and high costs of initial setup and/or individual experiments.

摘要空间转录组学技术旨在通过分析来自单个组织学载玻片的完整空间信息的整个转录组来推进基因表达研究。然而，空间转录组学的应用受到低分辨率，有限的转录覆盖率，复杂的程序，较差的可扩展性以及初始设置和/或单个实验的高成本的限制。

Seq-Scope repurposes the Illumina sequencing platform for high-resolution, high-content spatial transcriptome analysis, overcoming these limitations. It offers submicrometer resolution, high capture efficiency, rapid turnaround time and precise annotation of histopathology at a much lower cost than commercial alternatives.

Seq Scope将Illumina测序平台重新用于高分辨率，高内容的空间转录组分析，克服了这些限制。它以比商业替代品低得多的成本提供亚微米分辨率，高捕获效率，快速周转时间和组织病理学的精确注释。

This protocol details the implementation of Seq-Scope with an Illumina NovaSeq 6000 sequencing flow cell, allowing the profiling of multiple tissue sections in an area of 7 mm × 7 mm or larger. We describe the preparation of a fresh-frozen tissue section for both histological imaging and sequencing library preparation and provide a streamlined computational pipeline with comprehensive instructions to integrate histological and transcriptomic data for high-resolution spatial analysis.

该协议详细介绍了使用Illumina NovaSeq 6000测序流动池实现Seq Scope，可以在7 mm×7 mm或更大的区域内对多个组织切片进行分析。我们描述了用于组织学成像和测序文库制备的新鲜冷冻组织切片的制备，并提供了简化的计算流程，其中包含全面的说明，以整合组织学和转录组学数据以进行高分辨率空间分析。

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. Aside from array production and sequencing, which can be done in batches, tissue processing, library preparation and running the computational pipeline can be completed within 3 days by researchers with experience in molecular biology, histology and basic Unix skills.

这包括使用常规软件工具进行单细胞和空间分析，以及我们最近开发的无分割方法来分析亚微米分辨率的空间数据。除了可以分批完成的阵列生产和测序外，具有分子生物学，组织学和基本Unix技能经验的研究人员还可以在3天内完成组织处理，文库制备和运行计算管道。

Given its adaptability across various biological tissues, Seq-Scope establishes itself as an invaluable tool for.

鉴于其在各种生物组织中的适应性，Seq Scope将其确立为一种宝贵的工具。

The protocol repurposes an Illumina NovaSeq 6000 flow cell for spatial transcriptomics, generating high-resolution datasets and integrating a streamlined data-analysis pipeline.

该协议将Illumina NovaSeq 6000流通池用于空间转录组学，生成高分辨率数据集并集成简化的数据分析管道。

Leveraging commonly available Illumina equipment, the protocol offers researchers ultra-high, submicrometer resolution in spatial transcriptomics analysis with a comprehensive analytical pipeline, whole-transcriptome coverage, rapid turnaround, cost efficiency and versatility.

利用常用的Illumina设备，该协议为研究人员提供了超高，亚微米分辨率的空间转录组学分析，具有全面的分析流程，整个转录组覆盖范围，快速周转，成本效益和多功能性。

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Fig. 1: Overview of experimental procedures.Fig. 2: An overview of computational procedures.Fig. 3: Example outputs from the Illumina SAV application.Fig. 4: Flow cell disassembly and dicing.Fig. 5: Liquid handling in Seq-Scope chip surface treatment.Fig. 6: Tissue-freezing chamber.Fig. 7: Tissue sectioning and attachment.Fig.

图1：实验程序概述。图2：计算程序概述。图3:Illumina SAV应用程序的示例输出。图4：流通池拆卸和切割。图5:Seq Scope芯片表面处理中的液体处理。图6：组织冷冻室。图7：组织切片和附着。图。

8: Liquid- and chip-handling procedure.Fig. 9: Seq-Scope adapter frame and silicone isolator used in Part 3: library construction.Fig. 10: Conceptual representation of the NovaScope workflow based on the ‘Request’ option.Fig. 11: Spatial arrangement between tiles in the NovaSeq 6000 S4 flow cell.Fig.

8： 液体和碎屑处理程序。图9：第3部分：库结构中使用的Seq Scope适配器框架和硅胶隔离器。图10：基于“请求”选项的NovaScope工作流的概念表示。图11:NovaSeq 6000 S4流通池中瓷砖之间的空间排列。图。

12: An output sbcd image from Step 168 illustrates the distribution of all spatial barcodes (i.e., HDMIs).Fig. 13: An output smatch image from Step 172, showing the distribution of spatial barcodes (i.e., HDMIs) that matched to the 2nd-Seq FASTQ files.Fig. 14: An output sge image from Step 174, showing the distribution of spatial barcodes (HDMIs) that align to the reference genome.Fig.

12： 来自步骤168的输出sbcd图像说明了所有空间条形码（即HDMI）的分布。图13：来自步骤172的输出smatch图像，显示了与第二个Seq FASTQ文件匹配的空间条形码（即HDMIs）的分布。图14：来自步骤174的输出sge图像，显示了与参考基因组对齐的空间条形码（HDMI）的分布。图。

15: Comparison between sge (left) and aligned histology (right) images.Fig. 16: An exemplary multidimensional clustering result obtained by using Seurat.Fig. 17: An exemplary output image from Step 191, illustrating distinct zonation of hepatocellular factors.Fig. 18: An exemplary pixel-level output image from LDA-based analysis showing clear zonation of hepatocellular factors.Fig.

15： sge（左）和对齐组织学（右）图像之间的比较。图16：通过使用修拉获得的示例性多维聚类结果。图17：来自步骤191的示例性输出图像，示出了肝细胞因子的不同分区。图18：来自基于LDA的分析的示例性像素级输出图像，显示肝细胞因子的清晰分区。图。

19: An exemplary pixel-level output image from Seurat-based analysis showing hepatocellular and non-parenchymal cell factors.Fig. 20: An exemplary black-and-white segmentation image.Fig. 21: An exemplary overlay analysis to inspect the cell segmentation performance.Fig. 22: An exemplary multidimensional clustering result obtained by using Seurat, based on the single-cell data, segmented throu.

19： 来自基于修拉的分析的示例性像素级输出图像，显示肝细胞和非实质细胞因子。图20：示例性黑白分割图像。图21：用于检查单元分割性能的示例性叠加分析。图22：通过使用Seurat获得的基于单细胞数据的示例性多维聚类结果，通过分割。

Data availability

数据可用性

All source data described in this protocol are available online via public repositories, such as Zenodo and Deep Blue Data: minimal test run dataset (https://doi.org/10.5281/zenodo.10835761), shallow-sequenced liver dataset (https://doi.org/10.5281/zenodo.10840696), deep-sequenced liver dataset (https://doi.org/10.7302/tw62-4f97) and exemplary downstream analysis input (https://doi.org/10.5281/zenodo.10841777)..

该协议中描述的所有源数据都可以通过公共存储库在线获得，例如Zenodo和深蓝数据：最小测试运行数据集(https://doi.org/10.5281/zenodo.10835761)，浅测序肝脏数据集(https://doi.org/10.5281/zenodo.10840696)，深度测序肝脏数据集(https://doi.org/10.7302/tw62-4f97)和示例性下游分析输入(https://doi.org/10.5281/zenodo.10841777)。。

Code availability

代码可用性

All source code described in this protocol is available online via GitHub: NovaScope pipeline, v1.0.0 (https://github.com/seqscope/novascope) and NEDA, v1.0.0 (https://github.com/seqscope/NovaScope-exemplary-downstream-analysis).

该协议中描述的所有源代码都可以通过GitHub在线获得：NovaScope pipeline，v1.0.0(https://github.com/seqscope/novascope)和NEDA，v1.0.0(https://github.com/seqscope/NovaScope-exemplary-downstream-analysis)。

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Download referencesAcknowledgementsThe work was supported by the Taubman Institute Innovation Project (to H.M.K. and J.H.L.), the NIH (T32AG000114 to Y.K. and C.-S.C., K01AG061236 to M.K., R01DK118631 to G.J., R01AG079163 to M.K. and J.H.L., R01HG011031 and HHSN268201800002I to H.M.K.

下载参考文献致谢这项工作得到了陶伯曼研究所创新项目（致H.M.K.和J.H.L.），美国国立卫生研究院（T32AG000114致Y.K.和C.S.C.，K01AG061236致M.K.，R01DK118631致G.J.，R01AG079163致M.K.和J.H.L.，R01HG011031和HHSN268201800002I致H.M.K.的支持。

and R01DK133448 and UH3CA268091 to J.H.L.), Technology Transfer Talent Network (T3N) Postdoctoral Fellowship (to Y.K.) and a Gleen Foundation Core grant (to J.H.L.).Author informationAuthor notesThese authors contributed equally: Yongsung Kim, Weiqiu Cheng, Chun-Seok Cho.Authors and AffiliationsDepartment of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, USAYongsung Kim, Chun-Seok Cho, Yongha Hwang, Anna Park, Mitchell Schrank, Jer-En Hsu, Angelo Anacleto, Myungjin Kim & Jun Hee LeeDepartment of Biostatistics, University of Michigan School of Public Health, Ann Arbor, MI, USAWeiqiu Cheng, Yichen Si, Jingyue Xi & Hyun Min KangSpace Planning and Analysis, University of Michigan Medical School, Ann Arbor, MI, USAYongha HwangBiomedical Research Core Facilities Advanced Genomics Core, University of Michigan, Ann Arbor, MI, USAEllen Pedersen, Olivia I.

R01DK133448和UH3CA268091授予J.H.L.），技术转移人才网络（T3N）博士后奖学金（授予Y.K.）和Gleen基金会核心赠款（授予J.H.L.）。作者信息作者注意到这些作者做出了同样的贡献：金永成，郑伟秋，赵春熙。作者和所属机构密歇根大学医学院分子与整合生理学系，密歇根州安娜堡，美国金永成，赵春熙，黄永哈，安娜公园，米切尔·施兰克，徐杰恩，安杰洛·阿纳克托，金明进和李俊熙密歇根大学公共卫生学院生物统计学系，密歇根州安娜堡，美国密歇根大学魏邱城，宜城Si，Jingyue Xi＆Hyun Kangmin空间规划与分析，密歇根州安娜堡，美国密歇根大学黄永哈生物医学研究核心设施密歇根大学安娜堡分校高级基因组学核心N佩德森，奥利维亚一世。

Koues & Thomas WilsonDepartment of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USAThomas WilsonDepartment of Pathology, University of Michigan Medical School, Ann Arbor, MI, USAThomas WilsonDepartment of Genetics, Harvard Medical School, Boston, MA, USAChangHee LeeHuman Genetics Center, School of Public Health, University of Texas Health Science Center at Houston, Houston, TX, USAGoo JunAuthorsYongsung KimView author publicationsYou can also search for this author in.

Koues＆Thomas WilsonDepartment of Human Genetics，密歇根大学医学院，密歇根州安阿伯，美国密歇根大学医学院病理学系，密歇根州安阿伯，美国马萨诸塞州哈佛医学院遗传学系，马萨诸塞州波士顿，USAChangHee Leeheuman Genetics Center，公共卫生学院，德克萨斯大学休斯顿健康科学中心，USAGoo JunAuthorsYongsung KimView作者出版物您也可以在中搜索这位作者。

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PubMed Google ScholarContributionsY.K., C.-S.C., A.P., M.S., J.-E.H., M.K. and J.H.L. developed the experimental part of the protocol. W.C., Y.H., Y.S., J.X., A.A., C.L., G.J. and H.M.K. developed the computational part of the protocol. E.P., O.I.K., T.W., H.M.K. and J.H.L. developed the sequencing part of the protocol.

PubMed谷歌学术贡献。K、 ，C.-S.C.，A.P.，M.S.，J.-E.H.，M.K.和J.H.L.开发了该协议的实验部分。W、 C.，Y.H.，Y.S.，J.X.，A.A.，C.L.，G.J.和H.M.K.开发了协议的计算部分。E、 P.，O.I.K.，T.W.，H.M.K.和J.H.L.开发了该方案的测序部分。

Y.K., W.C., C.-S.C., H.M.K. and J.H.L. prepared the first draft. All authors revised, reviewed and approved the final version.Corresponding authorsCorrespondence to.

Y、 K.，W.C.，C.-S.C.，H.M.K.和J.H.L.编写了初稿。所有作者都修订，审查并批准了最终版本。通讯作者通讯。

Hyun Min Kang or Jun Hee Lee.Ethics declarations

康贤民或李俊熙。道德宣言

Competing interests

相互竞争的利益

H.M.K. owns stock in Regeneron Pharmaceuticals. J.H.L. is an inventor on a patent and pending patent applications related to Seq-Scope.

H、 M.K.拥有Regeneron Pharmaceuticals的股票。J、 H.L.是与Seq Scope相关的专利和未决专利申请的发明人。

Peer review

同行评审

Peer review information

同行评审信息

Nature Protocols thanks Moritz Gerstung and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

《自然协议》感谢Moritz Gerstung和另一位匿名审稿人对这项工作的同行评审做出的贡献。

Additional informationPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Related linksKey references using this protocolCho, C.-S. et al. Cell 184, 3559–3572.e22 (2021): https://doi.org/10.1016/j.cell.2021.05.010Xi, J.

。使用此协议的相关linksKey参考文献，C.-S.等人Cell 1843559–3572.e22（2021）：https://doi.org/10.1016/j.cell.2021.05.010Xi，J。

et al. Bioinform. Adv. 2, vbac061 (2022): https://doi.org/10.1093/bioadv/vbac061Si, Y. et al. Nat. Methods 21, 1843–1854 (2024): https://doi.org/10.1038/s41592-024-02415-2Do, T. H. et al. Sci. Immunol. 7, eabo2787 (2022): https://doi.org/10.1126/sciimmunol.abo2787Supplementary informationSupplementary Data 13D model (STL) file for the custom frame adapter described in the protocol.

等人。Bioinform。Adv.2，vbac061（2022年）：https://doi.org/10.1093/bioadv/vbac061Si，Y.等人，《自然方法》211843-1854（2024）：https://doi.org/10.1038/s41592-024-02415-2Do，T.H.等人，科学。免疫。7，eabo2787（2022年）：https://doi.org/10.1126/sciimmunol.abo2787Supplementary协议中描述的自定义帧适配器的信息补充数据13D模型（STL）文件。

The STL file can be used to fabricate the adapter in most 3D printing service centers. We printed the adapter on a Stratasys J850 by using the default white material.Supplementary Data 2Sketch drawing and specification of the custom silicone isolator (Grace Bio-Labs, cat. no. JTR25-A-1.0, RD501346).

STL文件可用于在大多数3D打印服务中心制造适配器。我们使用默认白色材料在Stratasys J850上打印了适配器。。

Information in this PDF is sufficient to reproduce the part with the same specifications applied by Grace Bio-Labs.Supplementary Data 3README file providing details on running the code and software applied in the protocolSupplementary Video 1NovaSeq 6000 S4 flow cell disassembly. The scalpel is used to separate the flow cell into its three main components.

此PDF中的信息足以复制具有Grace Bio Labs应用的相同规格的部件。补充数据3自述文件，提供有关运行协议补充视频1NovaSeq 6000 S4流通池反汇编中应用的代码和软件的详细信息。手术刀用于将流通池分离为三个主要组件。

This video demonstrates the entire procedure of the flow cell disassembly. During the demonstration, viewers may notice that a small piece was broken off the top layer of the flow cell. This layer is thin and, therefore, prone to breakage. However, as long as the broken piece is outside the imaging area described in Fig.

该视频演示了流通池拆卸的整个过程。在演示过程中，观众可能会注意到流动池顶层有一小块被折断。该层很薄，因此容易破裂。然而，只要碎片在Fig.描述的成像区域之外。

3d (B02–B10 and T02–T10), it does not interfere with subsequent procedures. Furthermore, even if the breakage damages some imagin.

3d（B02–B10和T02–T10），它不会干扰后续程序。此外，即使破损损坏了一些图像。

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