Nature Protocols最新文献

{"title":"Whole-organ spatial transcriptional analysis at cellular resolution using TRISCO.","authors":"Yue Li, Abigail Walton, Judith C Kreutzmann, Lea Lydolph Larsen, Ilse Eidhof, Jacob Lercke Skytte, Casper Gravesen Salinas, Urmas Roostalu, Jacob Hecksher-Sørensen, Shigeaki Kanatani, Per Uhlén","doi":"10.1038/s41596-026-01386-2","DOIUrl":"https://doi.org/10.1038/s41596-026-01386-2","url":null,"abstract":"<p><p>The emerging field of 3D histology centers on the molecular profiling of intact organs for a comprehensive understanding of biological systems. While current methods focus largely on protein visualization, techniques for whole-organ RNA transcript imaging remain underdeveloped. Here we present a detailed protocol for Tris buffer-mediated retention of in situ hybridization chain reaction signal in cleared organs (TRISCO), a method for single-cell RNA three-dimensional mapping across tissue volumes. By ensuring homogeneous and well-preserved labeling throughout the entire tissue, TRISCO has been successfully applied to several mouse organs, including the brain, lung, heart, kidney and spinal cord, as well as to rat and guinea pig brains. The protocol is straightforward, is adaptable to diverse laboratory setups and research questions and avoids complex instrumentation, specialized expertise or harsh chemical treatments that might limit its applicability. The workflow, which can be completed in 10-15 days, includes flexible stopping points for convenient scheduling and is suitable for users with expertise in light-sheet microscopy and animal handling.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148272442","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}

{"title":"High-resolution single-cell sequencing of trans-spliced mRNA.","authors":"Raúl O Cosentino, Zhibek Keneskhanova, Sandra Esser, T Nicolai Siegel","doi":"10.1038/s41596-026-01373-7","DOIUrl":"https://doi.org/10.1038/s41596-026-01373-7","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":18.4,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148265403","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}

{"title":"An immunocompetent bone marrow-on-a-chip model for studying human hematological malignancies and preclinical therapeutic screening.","authors":"Huishu Wang, Lunan Liu, Weiqiang Chen","doi":"10.1038/s41596-026-01387-1","DOIUrl":"https://doi.org/10.1038/s41596-026-01387-1","url":null,"abstract":"<p><p>The complex pathophysiology of leukemia and other bone marrow-related hematological malignancies underscores the critical need for immunocompetent bone marrow models that can accurately recapitulate key disease microenvironment and patient therapeutic responses. Existing bone marrow models often lack the fidelity required to recapitulate patient-specific pathophysiology or to emulate the intricate dynamics of in vivo immune responses. This limitation ultimately constrains their utility in critical areas such as leukemia immunotherapy development. Here, to overcome this challenge, we present a detailed protocol for building a 3D microfluidic human bone marrow-on-a-chip microphysiological system that recapitulates the anatomical organization and function of the native tissue. Its concentric three-compartment design replicates the marrow's spatial architecture, co-culturing stromal and hematopoietic cells within a vascularized niche to establish a physiologically relevant, immunocompetent microenvironment. To model patient-specific biology, the platform incorporates patient-derived samples, enabling the personalized evaluation of therapeutic interventions such as chimeric antigen receptor T cell therapy and chemotherapy. The platform supports multiplexed readouts through live imaging, immunofluorescence, cytokine profiling, flow cytometry and single-cell sequencing, enabling a comprehensive analysis of treatment response. The protocol requires ~7 d to establish the functional bone marrow chip, comprising microfluidic device fabrication (1 d), cell seeding (1 d) and progressive development of the bone marrow model (5 d). Therapeutic testing is conducted over a variable timeframe, typically ranging from 2 d to 14 d. The protocol is designed for researchers with basic experience in cell culture and fluorescence imaging,who have basic microfluidics fabrication and tissue culture experience.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-06-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148226898","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}

{"title":"Robust analytical methods for bis(monoacylglycero)phosphate profiling in health and disease.","authors":"Wentao Dong, Kwamina Nyame, Eshaan S Rawat, Uche N Medoh, Jian Xiong, Caio C Bonin, Hisham N Alsohybe, Hunter Y Liu, Sara Gomes, Tammy Shalit, Marianna Arnold, Frank Hsieh, Esther Sammler, Monther Abu-Remaileh","doi":"10.1038/s41596-026-01379-1","DOIUrl":"https://doi.org/10.1038/s41596-026-01379-1","url":null,"abstract":"<p><p>Bis(monoacylglycero)phosphates (BMPs), a distinct class of anionic phospholipids predominantly found in late endosomes and lysosomes, plays a pivotal role in supporting lysosomal functions and maintaining metabolic homeostasis. Dysregulation of BMPs is associated with an array of disorders, notably neurodegenerative diseases. However, the identification and quantitation of BMP remains difficult because of its structural similarity to its isomer, phosphatidylglycerol (PG), thus necessitating robust analytical methods for accurate and reliable BMP profiling. In this study, we present comprehensive liquid chromatography (LC)-tandem mass spectrometry (MS2) methodologies for the precise and systematic analysis of BMP species in biological samples. We detail LC/MS methods for both an untargeted Orbitrap mass spectrometer and a targeted triple quadrupole mass spectrometer. We use differences in hydrophobicity and structure to annotate BMPs and PGs on the basis of retention time and positive-mode MS2 fragmentation patterns, respectively. Because genetic ablation of the BMP synthase CLN5 leads to specific depletion of BMPs but not PGs, lipid extracts from CLN5 knockout and wild-type cells can be compared to confidently annotate BMPs when MS2 data are incomplete. Lipid extraction and preparation of samples for LC/MS takes ~4 h, unattended LC/MS instrument time depends on the number of samples and computer-based data analysis takes ~1 d. Altogether, this approach constitutes a robust method for BMP profiling and annotation, furthering research into health and disease.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148218475","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}

{"title":"Three-dimensional direct neuronal reprogramming for modeling Alzheimer's disease neuropathology.","authors":"Zhao Sun, Yoon Lee, Courtney K Walker, Celeste M Karch, Andrew S Yoo","doi":"10.1038/s41596-026-01388-0","DOIUrl":"https://doi.org/10.1038/s41596-026-01388-0","url":null,"abstract":"<p><p>Late-onset Alzheimer's disease (LOAD) accounts for over 95% of Alzheimer's disease (AD) cases. However, modeling the age-dependent neuropathological hallmarks of LOAD has remained a major challenge. We recently developed a patient-based, three-dimensional (3D) neuronal culture system that endogenously recapitulates key neuropathological features of AD, including extracellular Aβ deposition, tau dysregulation and spontaneous neuronal death. This platform uses high-efficiency, microRNA- and chromatin-based direct somatic reprogramming of fibroblasts from patients with autosomal dominant AD or LOAD to cortical neurons in 3D thin gel (3D-CNs) or self-assembled neuronal spheroids. Here we provide stepwise instructions for generating 3D-CNs and cortical spheroids via neuronal reprogramming of patient fibroblasts, along with methods for analyzing downstream AD neuropathology. Within 3 weeks, fibroblasts are reprogrammed into neurons under either thin-gel or spheroid conditions. By 4 weeks, key AD neuropathology, such as Aβ deposition, tau dysregulation, and neuronal death, can be detected in AD neurons. This system provides an age-relevant, patient-specific neuronal model for investigating molecular events underlying AD-associated neurodegeneration and evaluating compounds or gene targets in the context of potential personalized therapeutic interventions. Successful implementation of this protocol requires prior experience in mammalian cell culture, plasmid preparation, lentiviral production and standard confocal imaging techniques.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-06-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148218525","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}

{"title":"On-resin assembly of cysteine-reactive linkers for controlled site-selective antibody bioconjugation.","authors":"Sona Krajcovicova, Thomas Wharton, David R Spring","doi":"10.1038/s41596-026-01376-4","DOIUrl":"https://doi.org/10.1038/s41596-026-01376-4","url":null,"abstract":"<p><p>Antibody conjugates represent key advances in targeted biotherapeutics, combining the precision of antibodies with a range of functional payloads, including cytotoxic small molecules, enzymes and peptides, to achieve high selectivity, reduced off-target effects and an improved therapeutic window compared with conventional small-molecule drugs. Here we present a protocol for the solid-phase synthesis of tetra-divinylpyrimidine (tetraDVP) linkers, a modular and scalable strategy for generating cysteine-reactive linkers used in site-selective bioconjugation, enabling the rapid production of diverse antibody conjugates. This protocol describes a multistep synthetic workflow involving solution-phase intermediate synthesis, solid-phase assembly on resin, polyethylene glycol elongation, installation of divinylpyrimidine warheads, and mild cleavage conditions, enabling reproducible production of the final linker scaffold. TetraDVP linkers are designed to simultaneously rebridge all four interchain disulfide bonds of native IgG1 and IgG4 antibodies with a single molecule, enabling the controlled installation of functional payloads such as peptides, drugs or protein tags with controlled payload-to-antibody ratio. This approach provides the only reported strategy for conjugating a single payload to a native antibody without the need for chromatographic purification or genetic/glycan engineering. Compared with previous solution-phase routes, this solid-phase protocol improves yield, scalability and reproducibility while enabling rapid diversification of linker architecture. The complete procedure can be performed in ~2 weeks and provides a versatile platform for accessing tetraDVP linkers bearing a variety of functional handles for antibody conjugation.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148212029","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}

{"title":"Concurrent positional dynamics and activity mapping of DNA-binding proteins","authors":"Longfu Xu,&nbsp;Zhaowei Liu,&nbsp;Colleen Caldwell,&nbsp;Shyla Singh,&nbsp;Gijs J. L. Wuite","doi":"10.1038/s41596-026-01359-5","DOIUrl":"10.1038/s41596-026-01359-5","url":null,"abstract":"Single-molecule techniques have advanced our understanding of DNA–protein interactions, yet simultaneously tracking enzyme position on DNA, determining binding dynamics and resolving enzyme activity remained challenging. Here, we present a protocol for concurrent positional dynamics and activity mapping (C-DAM) of DNA-binding proteins that integrates optical tweezers and confocal fluorescence microscopy to achieve concurrent, high-resolution correlation of enzyme activity with its precise location on DNA. The protocol uses optical tweezers to track enzymes converting DNA between single- and double-stranded forms, measuring mechanical changes to resolve enzymatic activity at nanometer spatial and millisecond temporal resolution. This mechanical information is then correlated with simultaneously acquired, diffraction-limited fluorescence signals through ‘force-assisted imaging’, which pinpoints fluorescently labeled molecules relative to their enzymatic activity site. Unlike existing methods analyzing mechanical and fluorescence data separately, C-DAM leverages both techniques synergistically, providing direct, concurrent mapping of enzyme position, binding dynamics and catalytic activity. The protocol enables dissection of distinct functional states, rapid enzyme exchange events and location-specific behaviors, particularly for multifunctional proteins at dynamic single-stranded DNA (ssDNA)–double-stranded DNA (dsDNA) junctions. By correlating DNA and protein kymographs, the method distinguishes different binding modes, such as static binding to ssDNA versus diffusion on dsDNA, providing quantitative information on protein–DNA interactions. The protocol is broadly adaptable for investigating DNA-binding proteins and their complexes, with potential for future integration with techniques like Förster resonance energy transfer. Excluding sample preparation and instrument construction, data acquisition and analysis for a typical dataset takes 2–6 h and requires expertise in single-molecule biophysics, programming and data analysis. Concurrent positional dynamics and activity mapping (C-DAM) allows simultaneous tracking of protein location, binding dynamics and enzymatic activity on DNA substrates by integrating optical tweezers with confocal fluorescence microscopy.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 10","pages":"4816-4857"},"PeriodicalIF":18.4,"publicationDate":"2026-06-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148212020","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}

{"title":"Structural variant calling using Sniffles2","authors":"Luis F. Paulin,&nbsp;Hermann Romanek,&nbsp;Farhang Jaryani,&nbsp;Fritz J. Sedlazeck","doi":"10.1038/s41596-026-01362-w","DOIUrl":"10.1038/s41596-026-01362-w","url":null,"abstract":"Structural variants (SVs) are the most common nucleotide alteration per human genome compared to other variant types and have profound implications in evolution, diseases and regulation of genes. Sniffles2 (v2.6.3) is an open-source software for reliable detection of SVs ranging from 50 bp and upward across deletions, duplications, insertions, inversions and translocations, based on long-read sequencing. The tool is commonly used for various species and has identified multiple causative mutations in human diseases, key alleles in cancer progression. Here this protocol describes how to fully use Sniffles2 to accurately identify germline and mosaic SVs. The latter are low-variant allele fraction SVs (5–22% VAF) of parts of cells. We further provide detailed instruction for SV joint calling within Sniffles2 to enable tumor/normal calling or family trio analysis, which can scale up into large-scale population studies. Sniffles2 has been benchmarked and compared to currently available tools and has the highest precision while also being very fast, able to call SVs for a 40× human genome in ~34 CPU min (~8.5 min wall clock with the default four threads). It is designed to be easy to use for researchers with basic knowledge of Linux operating systems and the terminal. Sniffles2 is an open-source software for reliable detection of structural variants (50 bp and upward) from long-read sequencing. Sniffles2 is able to call variants over a wide variant allele fraction (5–100%) and across multiple samples (population).","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 10","pages":"4926-4938"},"PeriodicalIF":18.4,"publicationDate":"2026-06-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148205001","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}

{"title":"Label-free interferometry platform for drug response profiling of bioprinted tumor organoids at single-organoid resolution.","authors":"Bowen Wang, Peyton J Tebon, Thang L Nguyen, Graeme F Murray, Daniel C Guest, Jason Reed, Sara Sartini, Alice Soragni, Michael A Teitell","doi":"10.1038/s41596-026-01375-5","DOIUrl":"https://doi.org/10.1038/s41596-026-01375-5","url":null,"abstract":"<p><p>Organoids have become mainstay tools for drug discovery and personalized medicine. High-throughput imaging readouts for drug screening of tumor organoids are of particular interest as organoid-level quantification of responses provides insights into heterogeneity, which is relevant for predicting therapeutic efficacy and anticipating emergence of resistance. However, screening extracellular matrix (ECM)-embedded organoids remains technically challenging. Standard methods with manual cell seeding in thick ECM constructs impede imaging efficiency, whereas bulk endpoint assays are easy to implement but fail to resolve single-organoid-level drug response data. Here we present a protocol to bioprint cells within a temperature-sensitive ECM in thin, flat, square-shaped patterns in 96-well plates to establish three-dimensional (3D) cultures for efficient, high-throughput, time-resolved quantitative phase imaging at single-organoid resolution. Quantitative phase imaging of 3D-bioprinted organoids using high-speed live cell interferometry coupled with machine learning analyses enables label-free quantification of biomass, growth kinetics and drug response profiles of thousands of individual organoids per experiment. We demonstrate that the protocol can be leveraged to automatically generate plates containing thousands of organoids for high-throughput imaging and drug screening experiments, quantify growth and drug response heterogeneity, resolve rare phenotypes and identify predictive features of drug response profiles for fundamental studies and therapeutic decision-making. This protocol can be completed in 2 weeks or less and can be adapted to organoids derived from a variety of cell sources and to alternative screening paradigms. The protocol requires familiarity with coding and experience with 3D cell culture, optical assembly and software installation.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2026-06-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148163628","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}

{"title":"Celebrating 20 years of Nature Protocols","authors":"","doi":"10.1038/s41596-026-01377-3","DOIUrl":"10.1038/s41596-026-01377-3","url":null,"abstract":"Since Nature Protocols launched in 2006, protocol sharing has shifted from the margins to mainstream practice. Before the launch of online protocol journals, researchers relied on protocol textbooks, research papers and word of mouth. Now, researchers can choose from a range of platforms and journals to access and disseminate detailed step-by-step protocols.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"21 6","pages":"2397-2397"},"PeriodicalIF":16.0,"publicationDate":"2026-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.comhttps://www.nature.com/articles/s41596-026-01377-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148203161","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}