Nature Protocols最新文献

Multiplexed single-cell reduced representation bisulfite sequencing for genome-wide DNA methylation profiling. 用于全基因组DNA甲基化分析的多路单细胞减少亚硫酸酯测序。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-10-15 DOI: 10.1038/s41596-026-01452-9

Fang Liu, Jian Huang, Yanhua Zheng, Lina Fu, Xuanlu Wu, Ningning Zhao, Yunfei Wang, Andreas Gnirke, Alexander Meissner, Xiaoxue Wang, Fan Zhang, Hongcang Gu

{"title":"Multiplexed single-cell reduced representation bisulfite sequencing for genome-wide DNA methylation profiling.","authors":"Fang Liu, Jian Huang, Yanhua Zheng, Lina Fu, Xuanlu Wu, Ningning Zhao, Yunfei Wang, Andreas Gnirke, Alexander Meissner, Xiaoxue Wang, Fan Zhang, Hongcang Gu","doi":"10.1038/s41596-026-01452-9","DOIUrl":"https://doi.org/10.1038/s41596-026-01452-9","url":null,"abstract":"DNA methylation is a fundamental epigenetic modification that regulates gene expression, maintains genomic integrity and defines cellular identity. Conventional bulk sequencing methods, such as reduced-representation bisulfite sequencing (RRBS), have elucidated genome-wide methylation landscapes but do not reveal cell-to-cell variation, limiting insights into methylation heterogeneity during development, differentiation and disease. Here, we present multiplexed single-cell RRBS (Msc-RRBS), a streamlined and scalable protocol for parallel DNA methylation profiling of hundreds of individual cells. All enzymatic reactions, including cell lysis, restriction digestion, end repair and adapter ligation, are carried out in a single microwell, which eliminates the need for buffer exchange and minimizes DNA loss. Short, indexed adapters are used to efficiently remove adapter dimers and enable effective purification of library DNA by magnetic bead clean-up. The workflow supports the generation of sequencing libraries from single cells, pooled cells or sub-nanogram amounts of purified genomic DNA. The full workflow, from cell sorting to sequencing-ready libraries and data analysis, requires ~6 d for one or more 96-well plates. Msc-RRBS thus provides an efficient, reproducible and high-throughput approach for dissecting cell-to-cell DNA methylation heterogeneity across diverse biological and disease contexts. The experiments require basic molecular biology skills, and primary data processing can be performed by someone with limited bioinformatics training. More advanced analyses need additional computational expertise.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149195295","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

T cell immunomonitoring: a comparative analysis of traditional and novel methods to quantify and characterize human antigen-specific T cells. T细胞免疫监测：量化和表征人类抗原特异性T细胞的传统方法和新方法的比较分析。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-10-06 DOI: 10.1038/s41596-026-01453-8

Sandra Lazzaro, Geert Leroux-Roels, Sylvia Janetzki, Bruno Salaun, Laura Cook, Katka Franke, Isabel Poschke, Lukas Bunse, Marij J P Welters, Michael Fehlings, Sofie Pattyn, Gwenn Waerlop, Liselotte Brix, Noah J Tubo, Jeffrey J Molldrem, Viola Denninger, Wim J E van Esch, Nikolaj Pagh Kristensen, Yolanda D Mahnke, Florian Mair, Kilian Schober, Sven D Koch

{"title":"T cell immunomonitoring: a comparative analysis of traditional and novel methods to quantify and characterize human antigen-specific T cells.","authors":"Sandra Lazzaro, Geert Leroux-Roels, Sylvia Janetzki, Bruno Salaun, Laura Cook, Katka Franke, Isabel Poschke, Lukas Bunse, Marij J P Welters, Michael Fehlings, Sofie Pattyn, Gwenn Waerlop, Liselotte Brix, Noah J Tubo, Jeffrey J Molldrem, Viola Denninger, Wim J E van Esch, Nikolaj Pagh Kristensen, Yolanda D Mahnke, Florian Mair, Kilian Schober, Sven D Koch","doi":"10.1038/s41596-026-01453-8","DOIUrl":"https://doi.org/10.1038/s41596-026-01453-8","url":null,"abstract":"T cells are a key component of the adaptive immune system, playing a central role in the body's defense against pathogens and diseases. Antigen discovery and longitudinal immunomonitoring of antigen-specific T cell responses offer valuable insights into the activity and efficacy of immunotherapies and can guide development of new prophylactic or therapeutic treatments, such as vaccines and cellular therapies. Traditional immunomonitoring methods for T cell analysis, while foundational, offer limited insights because of the complexity and vast heterogeneity of antigen-specific T cells. Here, traditional and cutting-edge technologies for profiling antigen-specific T cells are reviewed, highlighting the evolution from low- to high-dimensional analyses. The principles, applications, benefits and limitations of each technique are outlined, and the need for assay harmonization and standardization is discussed. The importance of careful sample processing is also considered. This review highlights how immunomonitoring techniques can complement each other to maximize understanding of T cell-mediated immunity, a key aspect shaping immunotherapeutic development, now and in the future.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149112317","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

SIMworks-an integrated software suite for quality-controlled augmented quantitative structured illumination microscopy. simworks -一个集成的软件套件，用于质量控制增强定量结构照明显微镜。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-09-24 DOI: 10.1038/s41596-026-01444-9

Lior Pytowski, William Jandi, Adrian Henggeler, Wan Cho, Lothar Schermelleh, Fena Ochs

{"title":"SIMworks-an integrated software suite for quality-controlled augmented quantitative structured illumination microscopy.","authors":"Lior Pytowski, William Jandi, Adrian Henggeler, Wan Cho, Lothar Schermelleh, Fena Ochs","doi":"10.1038/s41596-026-01444-9","DOIUrl":"https://doi.org/10.1038/s41596-026-01444-9","url":null,"abstract":"Super-resolution structured illumination microscopy (SIM) is a powerful and versatile technique for investigating fundamental cell biological processes underlying development and disease. With the growing availability of commercial SIM systems, the emergence of advanced SIM modalities and increased access through imaging facilities, adoption of SIM across research laboratories is accelerating. However, fully leveraging the capabilities of SIM remains challenging, even for experienced microscopists, due to its susceptibility to imaging artifacts and the limited availability of accessible analysis pipelines. Here, to address these challenges, we developed SIMworks, a broadly compatible, integrated software platform designed to streamline quality control, processing and quantitative analysis of large-scale SIM datasets. Building on our previously established tools-SIMcheck, Chromagnon, ChaiN and SIMinspector-SIMworks provides a unified framework compatible with both custom and commercial SIM systems and modalities. Its modular architecture includes tools for calibration, alignment and data processing, as well as robust artifact detection and correction. These are complemented by segmentation and quantification modules for a broad range of cellular and nuclear structures, enabling automated, intensity-based analysis. Each module includes detailed guidance on interpretation and troubleshooting to support usability and reproducibility, enabling both new learners and experienced users to apply best practices in SIM imaging. The complete workflow can be performed within approximately half a day, depending on dataset size and complexity. SIMworks represents the first comprehensive, modular solution for SIM data processing, augmentation and quantitative analysis, lowering technical barriers and enhancing data reliability. By facilitating rigorous workflows, SIMworks promotes the broader integration of quantitative SIM imaging into biomedical research.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149039890","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

New approach methodologies: drug screening platform to identify antifibrotic compounds. 新方法方法：药物筛选平台识别抗纤维化化合物。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-09-24 DOI: 10.1038/s41596-026-01445-8

Hao Zhang, Lu Ren, Rihua Huang, Renke Tan, David E Solow-Cordero, Souhrid Mukherjee, Joseph C Wu

{"title":"New approach methodologies: drug screening platform to identify antifibrotic compounds.","authors":"Hao Zhang, Lu Ren, Rihua Huang, Renke Tan, David E Solow-Cordero, Souhrid Mukherjee, Joseph C Wu","doi":"10.1038/s41596-026-01445-8","DOIUrl":"https://doi.org/10.1038/s41596-026-01445-8","url":null,"abstract":"Fibrosis is characterized by excessive deposition of extracellular matrix, leading to organ stiffening and progressive dysfunction. Cardiac fibrosis, in particular, is a common feature in ischemic heart failure, genetic cardiomyopathies, diabetes mellitus and aging, which exacerbates cardiac dysfunction and ultimately contributes to increased morbidity and mortality. Despite its clinical burden and importance, there are currently no US Food and Drug Administration-approved drugs that specifically target cardiac fibrosis. This gap is largely due to the lack of reliable translational drug discovery platforms. To address this unmet clinical need, we present our new approach methodology drug screening platform for identifying novel compounds to treat cardiac fibrosis. This high-throughput screening system uses human reporter induced pluripotent stem (iPS) cell-derived cardiac fibroblasts to identify initial hits with potential antifibrotic effects, followed by counter-screening using iPS cell-derived cardiomyocytes to exclude compounds with potential cardiotoxicity. Compared to traditional primary cell-based approaches, our iPS cell-based workflow is robust, scalable and capable of screening ~5,000 compounds within 6-8 weeks. This protocol provides a versatile and efficient platform for phenotypic drug discovery with broad potential for translational applications in medicine.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149039869","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Tutorial: interrogating biology using organoid technologies. 教程：使用类器官技术询问生物学。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-09-24 DOI: 10.1038/s41596-026-01447-6

Jeroen M Bugter, Simon T Schafer, Roland Rad

引用次数: 0

Author Correction: Tracking-seq: a universal off-target detection approach for CRISPR-Cas genome editing. 作者更正：Tracking-seq：用于CRISPR-Cas基因组编辑的通用脱靶检测方法。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-09-22 DOI: 10.1038/s41596-026-01462-7

Runda Xu, Tingting Cong, Junsong Yuan, Xuancheng Chen, Yinqing Li, Xun Lan, Ming Zhu

引用次数: 0

Trace-n-Seq combines retrograde fluorescent tracing with sorting and single-cell sequencing of innervating peripheral neurons. Trace-n-Seq结合了逆行荧光示踪与排序和单细胞测序的支配周围神经元。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-09-21 DOI: 10.1038/s41596-026-01416-z

Vera Thiel, Manuel Mastel, Simon Renders, Jasper Panten, Katharina Bauer, Rene Jackstadt, Martin R Sprick, Andreas Trumpp

{"title":"Trace-n-Seq combines retrograde fluorescent tracing with sorting and single-cell sequencing of innervating peripheral neurons.","authors":"Vera Thiel, Manuel Mastel, Simon Renders, Jasper Panten, Katharina Bauer, Rene Jackstadt, Martin R Sprick, Andreas Trumpp","doi":"10.1038/s41596-026-01416-z","DOIUrl":"https://doi.org/10.1038/s41596-026-01416-z","url":null,"abstract":"Neuronal innervation plays a pivotal role in controlling tissue function during homeostasis and regeneration, as well as in pathological contexts such as inflammation, autoimmune disorders, fibrosis and cancer. A major obstacle in understanding neuron-tissue interactions at the molecular level is that neuronal cell bodies reside in peripheral ganglia often far outside the organ of interest and are therefore typically excluded in common single-cell datasets of tissues. Here we developed Trace-n-Seq to address this issue-a method that combines retrograde tracing with fluorescence-activated cell sorting and single-cell sequencing to molecularly profile individual neurons innervating healthy or diseased tissues. Trace-n-Seq can be used to explore the specific gene expression signatures of peripheral neurons at single-cell resolution. The method utilizes retrograde axonal tracing using Fast Blue, ganglia dissociation and fluorescence-activated cell sorting followed by single-cell RNA sequencing to enable the isolation and transcriptomic analysis of ganglia. Unlike bulk or whole-ganglia single-cell RNA sequencing without retrograde labeling, Trace-n-Seq allows the characterization of organ-specific neurons with single-cell resolution, eliminating background from uninvolved neurons and unrelated cell types located in the same ganglion. The procedure allows researchers to obtain molecular transcriptomes of tissue-innervating neurons and to study their plasticity, subtype identity and potential interactions with the tissue microenvironment. The complete Trace-n-Seq workflow can be performed in 2 weeks and is suitable for researchers with experience in molecular biology, neuronal tissue handling, sequencing techniques and bioinformatic analysis.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149017564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Tissue expansion mass spectrometry imaging (TEMI) for high-spatial-resolution multiomics molecular mapping. 组织膨胀质谱成像（TEMI）用于高空间分辨率多组学分子定位。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-09-21 DOI: 10.1038/s41596-026-01427-w

Hua Zhang, Lang Ding, Thao Duong, Kendra G Selby, Wan-Jo Lee, Amy Hu, Haiyan Lu, Paul W Tillberg, Meng Wang, Lingjun Li

{"title":"Tissue expansion mass spectrometry imaging (TEMI) for high-spatial-resolution multiomics molecular mapping.","authors":"Hua Zhang, Lang Ding, Thao Duong, Kendra G Selby, Wan-Jo Lee, Amy Hu, Haiyan Lu, Paul W Tillberg, Meng Wang, Lingjun Li","doi":"10.1038/s41596-026-01427-w","DOIUrl":"https://doi.org/10.1038/s41596-026-01427-w","url":null,"abstract":"High-spatial-resolution in situ mapping of biomolecules within tissue reveals critical insights into the complex molecular landscape and spatial organization of biological systems. Mass spectrometry imaging (MSI) is a powerful tool for spatially resolved molecular analysis of biological samples, with ongoing demand for improved spatial resolution. Tissue expansion combined with MSI (TEMI) is a recently developed approach that enables multiomics molecular mapping across various biological tissues with significantly improved spatial resolution. Unlike conventional methods that depend on instrument-based enhancements in spatial resolution, TEMI physically enlarges tissue samples via harsh-condition-free hydrogel expansion, achieving more than 3.5-fold increase in effective imaging resolution using standard MSI instrumentation. TEMI delivers single-cell spatial resolution in tissue samples and enables detection of biomolecular heterogeneity that remains uncharacterizable in unexpanded tissue using conventional MSI. Notably, TEMI supports high-spatial-resolution mapping of multiple biomolecular classes-including lipids, metabolites, N-glycans, peptides and proteins-within a single tissue sample. Here, we provide a detailed, step-by-step guide for TEMI, including hydrogel-based tissue expansion under mild conditions, cryosectioning of the expanded tissue-hydrogel sample, a comprehensive experimental workflow for multiomics TEMI on a single tissue section, data acquisition and visualization pipelines, as well as troubleshooting tips. Overall, we demonstrate that TEMI overcomes the long-standing spatial limitations of MSI without requiring hardware modifications, ensuring compatibility with existing MSI instruments and promoting broad accessibility and adoption within the research community.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149017602","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Head-fixed intravenous drug self-administration: a new frontier in addiction neuroscience. 头部固定静脉药物自我给药：成瘾神经科学的新前沿。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-09-17 DOI: 10.1038/s41596-026-01438-7

Kenichiro Negishi, Ginevra D'Ottavio, Yavin Shaham

引用次数: 0

Kit-based remote collection and isolation of human reprogrammable somatic cells for generation of induced pluripotent stem cells and cerebral organoids. 基于试剂盒的人类可重编程体细胞的远程采集和分离，用于诱导多能干细胞和脑类器官的生成。

IF 18.4 1区生物学

Nature Protocols Pub Date : 2026-09-16 DOI: 10.1038/s41596-026-01440-z

Iris Q Cheng, Juan F Ruiz, Evan K Casalino, Ce Zhang, Angeliki Louvi

{"title":"Kit-based remote collection and isolation of human reprogrammable somatic cells for generation of induced pluripotent stem cells and cerebral organoids.","authors":"Iris Q Cheng, Juan F Ruiz, Evan K Casalino, Ce Zhang, Angeliki Louvi","doi":"10.1038/s41596-026-01440-z","DOIUrl":"https://doi.org/10.1038/s41596-026-01440-z","url":null,"abstract":"Developing reproducible human disease models is a central goal of biomedical research and personalized medicine. Induced pluripotent stem (iPS) cell-derived models enable investigation of underlying cellular and molecular mechanisms in humans. However, obtaining reprogrammable somatic cells is a challenge, especially when patients have limited access to hospital and research facilities. Here we provide a step-by-step protocol for noninvasive isolation of human keratinocytes via trypsinization from scalp hair with intact follicles. The samples can be collected remotely by the donor, a family member or a healthcare provider using a kit and shipped long distance at ambient temperature. This offers a major advantage over widely used methods for iPS cell generation from other somatic cells, for example, dermal fibroblasts or peripheral blood mononuclear cells (PBMCs) (from invasive skin biopsies or blood collection), or renal epithelial cells (from urine). Keratinocytes can be reprogrammed to iPS cells that give rise to all three cell lineages, including the neural lineage, and used to generate cerebral organoids. The protocol yields iPS cells within 2 months of receiving the hair samples and requires basic familiarity with mammalian cell culture techniques without the need for specialized equipment. Generation of cerebral organoids from iPS cells requires 30-40 d and specialized equipment (an orbital shaker) for culture.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148982927","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0