Nature Protocols最新文献_第6页

Modular assembly of polyoxometalate clusters at the sub-1 nm scale. 模块化组装多金属酸氧酯簇在亚1纳米尺度。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-22 DOI: 10.1038/s41596-025-01212-1

Fenghua Zhang, Wenxiong Shi, Qingda Liu, Xun Wang

{"title":"Modular assembly of polyoxometalate clusters at the sub-1 nm scale.","authors":"Fenghua Zhang, Wenxiong Shi, Qingda Liu, Xun Wang","doi":"10.1038/s41596-025-01212-1","DOIUrl":"https://doi.org/10.1038/s41596-025-01212-1","url":null,"abstract":"Atomic-level manufacturing enables the bottom-up fabrication of nanomaterials with tailored structures and properties. Clusters with atomic precise structures can be used as superatom building blocks to construct superstructures with exceptional properties beyond their individual properties. However, the programmable and large-scale synthesis of cluster assemblies remains challenging. This protocol describes the detailed experimental procedures for the modular assembly of polyoxometalate (POM) clusters into subnanomaterials by programmable interactions under simple and mild conditions. In this approach different types of POM clusters (0.7-1.8 nm in size) are coated with quaternary ammonium or oleylamine ligands using either two-phase or solvothermal methods. The assembly process depends on the interactions between atom clusters, ligands and the reaction matrix, all of which can be modified to generate a library of subnanometer superstructures. The four intercluster connection modes are metal cation-induced coordinative connection, anion bridged covalent connection, synergistic noncovalent interaction and cluster-nucleus co-assembly. A library that includes single-cluster nanowires, clusterphenes and nanosheets with single-cluster thicknesses, can be prepared within 3-12 h. Owing to their ultrahigh surface atom ratio and electron delocalization, the resulting subnanometer POM assemblies with rich structural and compositional diversity exhibit excellent properties and application potential in terms of mechanics, catalysis and chirality. This procedure is suitable for users with prior expertise in the synthesis of inorganic and cluster-based nanomaterials.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144691030","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

Fluorescence-activated particle sorting for condensate purification. 用于冷凝水净化的荧光活化颗粒分选。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-18 DOI: 10.1038/s41596-025-01216-x

Annie Munier Godebert, Dominique Weil, Adham Safieddine

{"title":"Fluorescence-activated particle sorting for condensate purification.","authors":"Annie Munier Godebert, Dominique Weil, Adham Safieddine","doi":"10.1038/s41596-025-01216-x","DOIUrl":"https://doi.org/10.1038/s41596-025-01216-x","url":null,"abstract":"Condensates are receiving increasing attention because of their ability to organize subcellular space. In eukaryotes, nuclear condensates include nucleoli and paraspeckles, and cytoplasmic ones include P-bodies (PBs) and stress granules. One approach to investigate condensate biology is through analyzing their protein and RNA content. However, purifying condensates remains a challenge because of their densities being similar to various other organelles, and the absence of protein markers accumulating exclusively in them. These limitations, combined with the generally low number of condensates per cell, necessitate new approaches to tackle their purification. Here, we present a protocol describing fluorescence-activated particle sorting (FAPS) for purifying condensates. In brief, FAPS involves fluorescently labeling condensates to identify and isolate them from other cellular components via sorting. In this Protocol, we focus on PB purification, quality control and downstream characterization of PB protein and RNA contents. Although originally developed to purify PBs from human cell lines, FAPS can be adapted to various condensates across model organisms. The procedure requires knowledge in basic cell culture, molecular biology and flow cytometry and access to a fluorescence-activated cell sorter with sufficient sensitivity. It requires ~25-30 d, including a hands-on period of 15 d, to complete. In summary, FAPS allows the characterization of the content of diverse condensates across cell types and organisms.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144668049","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

Long-read RNA sequencing of transposable elements from single cells using CELLO-seq. 使用CELLO-seq对单细胞转座因子进行长读RNA测序。

IF 16 1区生物学

Nature Protocols Pub Date : 2025-07-16 DOI: 10.1038/s41596-025-01203-2

Sophie A Marlow, Lauryn A Deaville, Rebecca V Berrens

{"title":"Long-read RNA sequencing of transposable elements from single cells using CELLO-seq.","authors":"Sophie A Marlow, Lauryn A Deaville, Rebecca V Berrens","doi":"10.1038/s41596-025-01203-2","DOIUrl":"10.1038/s41596-025-01203-2","url":null,"abstract":"Transposable elements (TEs) form 50% of the mammalian genome sequence, and their expression contributes to processes from development to disease. Owing to the abundance and high sequence similarity of TEs, short-read single-cell sequencing methods promote ambiguous mapping to many almost-identical TE loci across the genome. Here we present a protocol for single-cell long-read RNA sequencing (CELLO-seq) to enable the mapping of TE-derived reads to unique genomic loci. CELLO-seq enables the error correction of long reads through the incorporation of long 50-nucleotide unique molecular identifiers and high polymerase chain reaction duplicate numbers. Taken together, these features of CELLO-seq enable the high-fidelity mapping of expression data to highly sequence-similar young TEs, as well as to gene and TE isoforms. This will enable in-depth exploration into the interaction of individual TEs and genes within single cells. This Protocol is designed to be accessible for users with experience in molecular biology and transcriptomic analysis and can be completed within a week from cell isolation through to quantification.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":16.0,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7617990/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144649922","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}

引用次数: 0

Developing chemical kinetic models for thermochemical applications. 开发热化学应用的化学动力学模型。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-16 DOI: 10.1038/s41596-025-01195-z

Marco Mehl, Matteo Pelucchi, Luna Pratali Maffei, Alessandro Stagni, Alberto Cuoci, Alessio Frassoldati, Eliseo Ranzi, Tiziano Faravelli

{"title":"Developing chemical kinetic models for thermochemical applications.","authors":"Marco Mehl, Matteo Pelucchi, Luna Pratali Maffei, Alessandro Stagni, Alberto Cuoci, Alessio Frassoldati, Eliseo Ranzi, Tiziano Faravelli","doi":"10.1038/s41596-025-01195-z","DOIUrl":"https://doi.org/10.1038/s41596-025-01195-z","url":null,"abstract":"A general procedure for the development of chemical kinetic models relevant to thermochemical applications (pyrolysis, gasification and combustion) is described. Here we present techniques that aim at producing models that are modular in structure, thoroughly validated, and applicable to a wide variety of conditions (generality), while balancing accuracy and computational burden. Starting from a core mechanism describing the pyrolysis and oxidation of light species, heavier compounds are added to the model in a hierarchical fashion, starting from archetypal species of each class of compounds. Using analogy rules derived from the archetypal species, a list of reactions and reaction rate parameters are compiled for molecules belonging to the classes of interest, obtaining detailed or semidetailed reaction mechanisms. The model is then validated using data available from the literature and/or novel experiments performed ad hoc. Depending on the applications of interest and on the size of the model, a mechanism reduction can be performed using a combination of lumping techniques and flux or sensitivity analyses. These procedures, although partially automated, still require some level of expert knowledge. The development of reaction rate rules and the identification of reaction pathways require indeed critical analysis and are most effective when the operator has previous experience in the field. A rigorously built mechanism, obeying the general principles presented here, provides high predictivity and permits extrapolating fuel behavior with greater confidence outside the range of validation conditions compared with models assembled from nonconsistently sourced submechanism from the literature, or based on limited datasets and empirical information.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-07-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144649921","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

scGPT: end-to-end protocol for fine-tuned retinal cell type annotation. scGPT：端到端协议微调视网膜细胞类型注释。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-15 DOI: 10.1038/s41596-025-01220-1

Shanli Ding, Jin Li, Rui Luo, Haotian Cui, Bo Wang, Rui Chen

{"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":"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.","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}

引用次数: 0

Scalable growth of vertical graphene nanosheets by thermal chemical vapor deposition. 垂直石墨烯纳米片的热化学气相沉积可扩展生长。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-10 DOI: 10.1038/s41596-025-01219-8

Quan Wu, Xixi Ji, Peilun Yu, Yuchao Cao, Zhenwei Li, Jie Yu, Yan Huang

{"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":"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.","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}

引用次数: 0

Assembly and delivery of large DNA via chromosome elimination in yeast. 酵母中通过染色体消除的大DNA的组装和传递。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-10 DOI: 10.1038/s41596-025-01214-z

Yuan Ma, Yao Xiong, Jiayu Xu, Hui Xu, Zongheng Fu, Guang-Rong Zhao, Yi Wu, Ying-Jin Yuan

{"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":"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.","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}

引用次数: 0

A systematic protocol to assess life cycle environmental and health impacts. 评估生命周期对环境和健康影响的系统规程。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-09 DOI: 10.1038/s41596-025-01206-z

Sabrina Spatari

引用次数: 0

Performing life cycle impact assessment with the midpoint and endpoint method ReCiPe. 使用中点和端点方法进行生命周期影响评估。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-09 DOI: 10.1038/s41596-025-01207-y

Rosalie van Zelm, Thomas Hennequin, Mark A J Huijbregts

{"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":"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.","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}

引用次数: 0

Nano3P-seq: charting the coding and noncoding transcriptome at single-molecule resolution. Nano3P-seq：在单分子分辨率上绘制编码和非编码转录组。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-07-08 DOI: 10.1038/s41596-025-01205-0

Oguzhan Begik, Leszek P Pryszcz, Adnan Muhammad Niazi, Eivind Valen, Eva Maria Novoa

{"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":"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.","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}

引用次数: 0