Nature Protocols最新文献

{"title":"scGPT: end-to-end protocol for fine-tuned retinal cell type annotation.","authors":"Shanli Ding, Jin Li, Rui Luo, Haotian Cui, Bo Wang, Rui Chen","doi":"10.1038/s41596-025-01220-1","DOIUrl":"https://doi.org/10.1038/s41596-025-01220-1","url":null,"abstract":"<p><p>Single-cell research faces challenges in accurately annotating cell types at high resolution, especially when dealing with large-scale datasets and rare cell populations. To address this, foundation models such as single-cell generative pretrained transformer (scGPT) offer flexible, scalable solutions by leveraging transformer-based architectures. Here we provide a comprehensive guide to fine-tuning scGPT for cell-type classification in single-cell RNA sequencing data. We demonstrate how to fine-tune scGPT on a custom retina dataset, highlighting the model's efficiency in handling complex data and improving annotation accuracy achieving 99.5% F1-score. This protocol automates key steps, including data preprocessing, model fine-tuning and evaluation. This protocol enables researchers to efficiently deploy scGPT for their own datasets. The provided tools, including a command-line script and Jupyter Notebook, simplify the customization and exploration of the model, proposing an accessible workflow for users with minimal Python and Linux knowledge. The protocol offers an off-the-shell solution of high-precision cell-type annotation using scGPT for researchers with intermediate bioinformatics. The source code and example datasets are publicly available on GitHub and Zenodo.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144642982","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":"Scalable growth of vertical graphene nanosheets by thermal chemical vapor deposition.","authors":"Quan Wu, Xixi Ji, Peilun Yu, Yuchao Cao, Zhenwei Li, Jie Yu, Yan Huang","doi":"10.1038/s41596-025-01219-8","DOIUrl":"https://doi.org/10.1038/s41596-025-01219-8","url":null,"abstract":"<p><p>Vertical graphene nanosheets (VGSs) are a kind of graphene materials, which retain the inherent advantages of graphene and effectively overcome the stacking bottleneck displayed by traditional graphene. The scalable production of VGSs may help the development of devices such as field-effect transistors, sensors, biomedical materials, electrochemical energy storage, thermal conductive materials and catalyst supports. The thermal chemical vapor deposition (CVD) approach has become a mature, efficient and highly valuable industrial strategy for VGSs fabrication. This technique imposes no restrictions on the morphology and size of the substrate and has high yield and low equipment cost, making it suitable for scalable industrial applications. Here we detail the step-by-step instructions for growing VGSs on a variety of common substrates such as carbon nanofibers, carbon fibers and Si particles using the thermal CVD approach. The scalability of thermal CVD could help advance the development of industrial applications of VGSs composite materials. The procedure requires a total of 136 h and 45 min to successfully produce VGSs on C and Si substrates, followed by a comprehensive characterization of the nanosheets. The procedure is suitable for users with expertise in chemistry or materials science.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144608866","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":"Assembly and delivery of large DNA via chromosome elimination in yeast.","authors":"Yuan Ma, Yao Xiong, Jiayu Xu, Hui Xu, Zongheng Fu, Guang-Rong Zhao, Yi Wu, Ying-Jin Yuan","doi":"10.1038/s41596-025-01214-z","DOIUrl":"https://doi.org/10.1038/s41596-025-01214-z","url":null,"abstract":"<p><p>Manipulation of large-scale genetic information provides a powerful approach to deciphering and engineering complex biological functions. However, the manipulation of large DNA, such as assembly and delivery, remains complex and difficult. Here we describe the experimental design strategy and protocol for a chromosome elimination-mediated large DNA assembly and delivery method (HAnDy), which enables efficient Mb-scale DNA assembly and delivery in yeast conveniently. This protocol is divided into three parts: (1) CRISPR-Cas9-mediated elimination of chromosome, which includes design and integration of a synthetic single-guide RNA (sgRNA) site near the centromere, activation of chromosome elimination by mating, and verification of the chromosome elimination. It can be used to eliminate multiple chromosomes, achieving haploidization in yeast. (2) Haploidization-mediated DNA assembly, which includes the design and construction of initial assembly strains, DNA assembly by programmed haploidization and verification of the assembled clones. (3) Haploidization-mediated DNA delivery, which includes the design and construction of inducible haploidization strains, DNA delivery by programmed haploidization and verification of the delivered clones. Users can utilize this protocol entirely or selectively depending on their needs. With the use of this protocol, it takes 10 d to achieve chromosome elimination and 7-11 d to achieve a standard cycle of haploidization-mediated DNA assembly or delivery. This protocol provides an efficient approach that is useful for the elimination, assembly and delivery of large DNA in yeast, requiring basic molecular biology skills.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144608865","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":"A systematic protocol to assess life cycle environmental and health impacts.","authors":"Sabrina Spatari","doi":"10.1038/s41596-025-01206-z","DOIUrl":"https://doi.org/10.1038/s41596-025-01206-z","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144600982","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":"Performing life cycle impact assessment with the midpoint and endpoint method ReCiPe.","authors":"Rosalie van Zelm, Thomas Hennequin, Mark A J Huijbregts","doi":"10.1038/s41596-025-01207-y","DOIUrl":"https://doi.org/10.1038/s41596-025-01207-y","url":null,"abstract":"<p><p>Life cycle assessment (LCA) is a method to understand and reduce the environmental impact of products over their life cycle. Although general guidelines to perform LCAs are available, specific recommendations on performing and reporting the life cycle impact assessment (LCIA) step in a standardized way are lacking. This lack can lead to incomplete results, followed by misinterpretation. In the LCIA step, the magnitude and significance of the potential environmental impacts are quantified and evaluated. Here, we describe how to systematically perform and report the LCIA step, identify the most meaningful LCA results and check their robustness. To develop the procedure, we used the widely applied LCIA methodology ReCiPe, which includes so-called characterization factors that express the environmental impact per unit of emission or extraction for 18 midpoint categories, such as global warming and acidification, and three endpoint categories (human health damage, ecosystem damage and resource scarcity). The characterization factors are developed for three perspectives, addressing inherent value choices in the calculation models. To demonstrate its use, our method was applied to a passenger car tire case study. We argue for the inclusion of all three endpoint categories and all three perspectives in the initial assessment. Furthermore, we recommend including a midpoint-to-endpoint contribution analysis on the impact results to identify the most important midpoint categories. Being comprehensive on the LCIA results will lead to a clear, distilled message to stakeholders to decrease environmental impacts, without unintended burden shifting across the supply chain or between different environmental impacts.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144600983","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":"Nano3P-seq: charting the coding and noncoding transcriptome at single-molecule resolution.","authors":"Oguzhan Begik, Leszek P Pryszcz, Adnan Muhammad Niazi, Eivind Valen, Eva Maria Novoa","doi":"10.1038/s41596-025-01205-0","DOIUrl":"https://doi.org/10.1038/s41596-025-01205-0","url":null,"abstract":"<p><p>RNA polyadenylation is crucial for RNA maturation, stability and function, with poly(A) tail lengths significantly influencing mRNA translation, efficiency and decay. Here, we provide a step-by-step protocol to perform Nanopore 3' end-capture sequencing (Nano3P-seq), a nanopore-based cDNA sequencing method to simultaneously capture RNA abundances and tail-composition and tail-length estimates at single-molecule resolution. Taking advantage of a template switching-based protocol, Nano3P-seq can sequence any RNA-derived molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or RNA adapter ligation. We provide an updated Nano3P-seq protocol that is compatible with R10.4 flow cells, as well as compatible software for poly(A) tail length and content prediction, which we term 'PolyTailor'. We demonstrate that PolyTailor provides accurate estimates of transcript abundances and tail lengths and composition, while capturing both coding and noncoding RNA biotypes, including mRNAs, small nucleolar RNAs and ribosomal RNAs. Nano3P-seq can be applied to RNA samples prepared by using different methods (e.g., poly(A)-selected, ribodepleted or total RNA) and can be completed in 1 day. The protocol requires experience in molecular biology techniques and nanopore sequencing library preparation and basic knowledge of Linux Bash syntax and R programming. This protocol makes Nano3P-seq accessible and easy to implement by future users aiming to study the tail dynamics and heterogeneity of both coding and noncoding transcriptomes in a comprehensive and reproducible manner.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144591825","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":"Detection of intramolecular protein dynamics on nanosecond-to-microsecond timescales by nanoparticle-assisted NMR spin relaxation (NASR).","authors":"Xinyao Xiang, Alexandar L Hansen, Lei Bruschweiler-Li, Rafael Brüschweiler, Mouzhe Xie","doi":"10.1038/s41596-025-01177-1","DOIUrl":"https://doi.org/10.1038/s41596-025-01177-1","url":null,"abstract":"<p><p>Proteins under physiological conditions have an intrinsically dynamic nature; they sample a multitude of different conformational substates that allow them to perform their biological functions. Protein motions can take place on a wide range of timescales. Although there are many different NMR experiments with sensitivity to different time windows, it has proven difficult to measure intramolecular motions that happen in the nanosecond-to-microsecond regime. Nanoparticle-assisted NMR spin relaxation (NASR) has recently been introduced to overcome this long-standing challenge. When colloidal nanoparticles are added to proteins in solution, the effective global tumbling of the protein molecules slows down, whereas the internal motions remain essentially unperturbed. NASR extends the protein dynamics observation window from picoseconds all the way into the microsecond range. In this protocol, the NASR effect is realized by using commercially available silica nanoparticles, and NMR measurements are acquired by using a standard high-field solution NMR spectrometer. NASR data analysis is shown to be straightforward. We demonstrate NASR by detecting sub-microsecond dynamics in the Switch I and II regions of oncogenic human KRAS and in the Loop I region of bacterial colicin-immunity protein Im7, among other protein constructs. When an isotope-labeled protein sample is available, this protocol can be executed in 2-5 d, including sample preparation, NMR experiments and data processing and analysis, to uncover potentially functionally important intramolecular dynamics at atomic resolution on timescales that are several orders of magnitude slower than what conventional spin relaxation experiments can observe.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144591824","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 robotic structures fabricated and powered entirely with proteins.","authors":"Haiyang Jia, Huan Sun, Johannes Flommersfeld, Wentao Shi, Frank Siedler, Petra Schwille","doi":"10.1038/s41596-025-01186-0","DOIUrl":"https://doi.org/10.1038/s41596-025-01186-0","url":null,"abstract":"<p><p>Assembling and upscaling biomolecular activity to perform work in man-made devices is a challenge in synthetic biology. Here we report the step-by-step process to construct fully protein-based micro-three-dimensional (3D) printed robotic structures, which are coated with and actuated by a minimal actomyosin cortex. This approach can be used to program self-powered soft robots assembled from multiple biomolecular modules, devising biophysical assays to quantify active forces produced in 3D and engineering smart 3D microchips for synthetic cell assembly. The procedure covers the establishment of 3D printing microstructures from protein materials, the assembly of actomyosin-based active coatings and the robotic structure design and characterization. The detailed step-by-step instructions will guide scientists in replicating the preparation procedures, facilitating the adoption of biomolecular microrobots and the development of 3D protein-based robotic technology and their applications. The procedure is suited for users with expertise in biomaterials and requires 15 d to complete.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144554002","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":"Expansion and fluctuations-enhanced microscopy for nanoscale molecular profiling of cells and tissues.","authors":"Dominik Kylies, Hannah S Heil, Arturo G Vesga, Mario Del Rosario, Maria Schwerk, Malte Kuehl, Milagros N Wong, Victor G Puelles, Ricardo Henriques","doi":"10.1038/s41596-025-01178-0","DOIUrl":"https://doi.org/10.1038/s41596-025-01178-0","url":null,"abstract":"<p><p>Advances in super-resolution microscopy enable the molecular profiling of cells and tissues at the nanoscale level, surpassing the diffraction limit of conventional light microscopy. However, super-resolution techniques typically require access to expensive specialized equipment and extensive training, limiting their broad applicability. Here we provide a detailed protocol for combining expansion microscopy with enhanced super-resolution radial fluctuations analysis to achieve nanoscale resolution using conventional microscopes. Expansion microscopy physically enlarges the sample, while enhanced super-resolution radial fluctuations computationally enhances the image resolution by analyzing fluorescence fluctuations over time. By combining both, we achieve images with a resolution of 25 nm in combination with diffraction-limited microscopes. Our step-by-step instructions include the expansion of cells and tissue samples, the optimization of multispectral microscopy parameters and the implementation of quality control metrics to minimize artifacts. We further cover the use of quantitative tools such as NanoJ-SQUIRREL, which enable the assessment of resolution improvements and image fidelity. We discuss key considerations for each stage, including sample preparation, image acquisition, computational processing and downstream analysis. Potential pitfalls and troubleshooting strategies are also addressed. This protocol can be used for imaging a variety of sample types with multiple fluorescent labels. With nanoscale spatial resolution and molecular specificity, expansion-enhanced super-resolution radial fluctuations microscopy provides a flexible, accessible approach for investigating cellular ultrastructure, protein localization and interaction networks, suitable for applications in cell biology, histopathology and biomedical research. The procedure requires 3-4 d to complete, involving ~7-9 h of total bench, imaging and processing time and only requires basic expertise in tissue handling, molecular and cell biology, and microscopy.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144554001","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":"A graphic and command line protocol for quick and accurate comparisons of protein and nucleic acid structures with US-align.","authors":"Chengxin Zhang, Lydia Freddolino, Yang Zhang","doi":"10.1038/s41596-025-01189-x","DOIUrl":"https://doi.org/10.1038/s41596-025-01189-x","url":null,"abstract":"<p><p>With the success of structural biology and the advancements in deep-learning-based structure predictions, rapid and accurate structural comparisons among macromolecular structures have become increasingly important in structural bioinformatics. US-align is a highly efficient, versatile, open-source program for sequential and nonsequential structure comparisons of proteins, RNAs and DNAs in pairwise and multiple alignment forms and applicable to both monomeric and multimeric complex structures. The core algorithm of US-align is built on a highly optimized, iterative superimposition and dynamic programming alignment process, guided with a unified and sequence length-independent scoring function, TM-score. The unique design of US-align not only ensures its high accuracy and speed compared with other state-of-the-art methods designed for specific alignment tasks but also makes it the only protocol that can be applied to multiple alignment tasks and allow a structural comparison across different molecular types, the latter of which is critical for template-based heteromolecular structure prediction and function annotations. Here we describe how to install and effectively utilize US-align as a command line tool, as an online web server, and as a plugin to commonly used molecular graphic systems such as PyMOL. US-align installation takes a few minutes to setup, while the actual alignment implementation can be completed typically within 1 s.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144554000","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}