Nature Protocols最新文献

{"title":"Retina-specific laminin-based generation of photoreceptor progenitors from human pluripotent stem cells under xeno-free and chemically defined conditions.","authors":"Wei Sheng Tan, Yixin Lai, Yingying Chung, Swarnaseetha Adusumalli, Xin Yi Lee, Karl Tryggvason, Hwee Goon Tay","doi":"10.1038/s41596-025-01142-y","DOIUrl":"https://doi.org/10.1038/s41596-025-01142-y","url":null,"abstract":"<p><p>Photoreceptor cell replacement therapy for retinal degenerative diseases is a promising approach. Presently, most protocols aimed at generating clinically safe and functional cells for retinal diseases face challenges such as low efficiency, poor reproducibility, and time-consuming and complex procedures. These could be due to the dependency on animal-derived components in cell culture media and substrates that support the cell differentiation process. Such conditions are poorly defined chemically, which could affect the robustness of the method and hinder clinical translation of cell therapy in retinal diseases. Here, we describe a simple protocol that is xenogen free and chemically defined to differentiate human embryonic stem cells to photoreceptor progenitors. Human recombinant extracellular matrix laminin 523 and 521 isoforms were used to mimic the inter-photoreceptor matrix niche environment to promote the retinal cell differentiation process. This was also accomplished by the unique combination of two cell differentiation media that recapitulates the retinal development signaling processes. In comparison to other protocols, our protocol does not require any mechanical dissection, which can be technically subjective and tedious. Our directed differentiation method generates photoreceptor progenitors that express ~17% cone-rod homeobox (CRX) transcript based on single-cell transcriptomic analyses by day 32. These day 32 photoreceptor progenitors can be cryopreserved and still maintain high cell viability after thawing for cell transplantation. This protocol can be easily reproduced and performed by researchers with basic cell culture experience, which is particularly important for retinal research progress and clinical cell manufacturing in a Good Manufacturing Practice facility.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143502413","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":"Circular Vectors as an efficient, fully synthetic, cell-free approach for preparing small circular DNA as a plasmid substitute for guide RNA expression in CRISPR-Cas9 genome editing.","authors":"Roman Teo Oliynyk, George M Church","doi":"10.1038/s41596-024-01138-0","DOIUrl":"https://doi.org/10.1038/s41596-024-01138-0","url":null,"abstract":"<p><p>Robust expression of guide RNA (gRNA) is essential for successful implementation of CRISPR-Cas9 genome-editing methods. The gRNA components, such as an RNA polymerase promoter followed by the gRNA coding sequence and an RNA polymerase terminator sequence, and the Cas9 protein are expressed either via an all-in-one plasmid or separate dedicated plasmids. The preparation of such plasmids involves a laborious multi-day process of DNA assembly, bacterial cloning, validation, purification and sequencing. Our Circular Vector (CV) protocol introduces an efficient, fully synthetic, cell-free approach for preparing gRNA expression templates suitable for transfection, marking a significant advancement over traditional plasmid-based approaches. This protocol consists of the circularization and purification of linear double-stranded DNA (dsDNA) containing gRNA expression elements into compact, bacterial-backbone-free circular DNA expression vectors in as little as 3 h. We provide a guide to the design of the dsDNA template coding for gRNA elements for CRISPR-Cas9 base and prime editing, along with step-by-step instructions for the efficient preparation of gRNA-expressing CVs. In addition to rapid preparation, CVs created via this protocol offer several key advantages: a compact size, absence of a bacterial backbone, absence of bacterial endotoxins and no contamination by bacterial RNA or DNA fragments. These features make gRNA-expressing CVs a superior choice over plasmid-based gRNA expression templates.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143492717","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":"Linking single-cell transcriptomes with secretion using SEC-seq.","authors":"Justin Langerman, Sevana Baghdasarian, Rene Yu-Hong Cheng, Richard G James, Kathrin Plath, Dino Di Carlo","doi":"10.1038/s41596-024-01112-w","DOIUrl":"https://doi.org/10.1038/s41596-024-01112-w","url":null,"abstract":"<p><p>Cells secrete numerous proteins and other biomolecules into their surroundings to achieve critical functions-from communicating with other cells to blocking the activity of pathogens. Secretion of cytokines, growth factors, extracellular vesicles and even recombinant biologic drugs defines the therapeutic potency of many cell therapies. However, gene expression states that drive specific secretory phenotypes are largely unknown. We provide a protocol that enables the secretion amount of a target protein encoded (SEC) by oligonucleotide barcodes to be linked with transcriptional sequencing (seq) for thousands of single cells. SEC-seq leverages microscale hydrogel particles called Nanovials to isolate cells and capture their secretions in close proximity, oligonucleotide-labeled antibodies to tag secretions on Nanovials and flow cytometry and single-cell RNA-sequencing (scRNA-seq) platforms for readout. Cells on Nanovials can be sorted on the basis of viability, secretion amount or other surface markers without fixation or permeabilization, and cell- and secretion-containing Nanovials are directly introduced into microfluidic droplets-in-oil emulsions for single-cell barcoding of cell transcriptomes and secretions. We have used SEC-seq to link T cell receptor sequences to the relative amount of associated cytokine secretions, surface marker gene expression with a highly secreting and potential regenerative population of mesenchymal stromal cells and the transcriptome with high immunoglobulin secretion from plasma cells. Nanovial modification and cell loading takes <4 h, and once the desired incubation time is over, staining, cell sorting and emulsion generation for scRNA-seq can also be completed in <4 h. Compared to related techniques that link secretions to a cell's surface, SEC-seq provides a general solution across any secretion target because of the ease with which biotinylated Nanovials can be modified. By linking gene expression and secretory strength, SEC-seq can expand our understanding of cell secretion, how it is regulated and how it can be engineered to make better therapies.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143468606","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":"Measuring plasma membrane fluidity using confocal microscopy.","authors":"Pablo Carravilla, Luca Andronico, Jan Schlegel, Yagmur B Urem, Ellen Sjule, Franziska Ragaller, Florian Weber, Cenk O Gurdap, Yavuz Ascioglu, Taras Sych, Joseph Lorent, Erdinc Sezgin","doi":"10.1038/s41596-024-01122-8","DOIUrl":"https://doi.org/10.1038/s41596-024-01122-8","url":null,"abstract":"<p><p>Membrane fluidity is a crucial parameter for cellular physiology. Recent evidence suggests that fluidity varies between cell types and states and in diseases. As membrane fluidity has gradually become an important consideration in cell biology and biomedicine, it is essential to have reliable and quantitative ways to measure it in cells. In the past decade, there has been substantial progress both in chemical probes and in imaging tools to make membrane fluidity measurements easier and more reliable. We have recently established a robust pipeline, using confocal imaging and new environment-sensitive probes, that has been successfully used for several studies. Here we present our detailed protocol for membrane fluidity measurement, from labeling to imaging and image analysis. The protocol takes ~4 h and requires basic expertise in cell culture, wet lab and microscopy.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143458667","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":"Construction of RNA reference materials for improving the quantification of transcriptomic data.","authors":"Ying Yu, Wanwan Hou, Qingwang Chen, Xiaorou Guo, Leqing Sang, Hao Xue, Duo Wang, Jinming Li, Xiang Fang, Rui Zhang, Lianhua Dong, Leming Shi, Yuanting Zheng","doi":"10.1038/s41596-024-01111-x","DOIUrl":"https://doi.org/10.1038/s41596-024-01111-x","url":null,"abstract":"<p><p>RNA reference materials and their corresponding reference datasets act as the 'ground truth' for the normalization of experimental values and are indispensable tools for reliably measuring intrinsically small differences in RNA-sequencing data, such as those between molecular subtypes of diseases in clinical samples. However, the variability in 'absolute' expression profiles measured across different batches, methods or platforms limits the use of conventional RNA reference datasets. We recently proposed a ratio-based method for constructing reference datasets. The ratio for a gene is defined as the normalized expression levels between two sample groups and produces more reliable values than the 'absolute' values obtained across diverse transcriptomic technologies and batches. Our gene ratios have been used for the successful generation of omics-wide reference datasets. Here, we describe a step-by-step process for establishing RNA reference materials and reference datasets, covering three stages: (1) reference materials, including material preparation, homogeneity testing and stability testing; (2) ratio-based reference datasets, including characterization, uncertainty estimation and orthogonal validation; and (3) applications, including definition of performance metrics, performing proficiency tests and diagnosing and correcting batch effects. This approach established the Quartet RNA reference materials and reference datasets (chinese-quartet.org) that have been approved as the first suite of nationally certified RNA reference materials by China's State Administration for Market Regulation. The protocol can be utilized to establish and apply reference materials to improve RNA-sequencing data quality in diverse clinical settings. The procedure can be completed in 2 d and requires expertise in molecular biology and bioinformatics.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143449527","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":"Synthesis of bicyclo[3.1.1]heptanes, meta-substituted arene isosteres, from [3.1.1]propellane.","authors":"Bhaskar Paul, Ayan Dasgupta, Nils Frank, Jeremy Nugent, Edward A Anderson","doi":"10.1038/s41596-024-01109-5","DOIUrl":"https://doi.org/10.1038/s41596-024-01109-5","url":null,"abstract":"<p><p>The use of saturated small-ring bridged hydrocarbons as bioisosteres for aromatic rings has become a popular tactic in drug discovery. Perhaps the best known of such hydrocarbons is bicyclo[1.1.1]pentane, for which the angle between the exit vectors of the bridgehead substituents is identical to that of a para-substituted arene (180°). The development of meta-arene (bio)isosteres is much less explored due to the challenge of identifying an accurate geometric mimic (substituent exit vector angle ~120°, dihedral angle ~0°). To address this, we recently reported straightforward access to bicyclo[3.1.1]heptanes (BCHeps), which exactly meet these geometric properties, via radical ring-opening reactions of [3.1.1]propellane. This required the development of a scalable synthesis of [3.1.1]propellane, as well as the implementation of various ring-opening reactions and derivatizations. Here we describe methodology for a multigram scale synthesis of [3.1.1]propellane in five steps from commercially available ethyl 4-chlorobutanoate, which proceeds in an overall yield of 26-37%. We also describe the functionalization of [3.1.1]propellane to three key classes of BCHep iodides by photocatalyzed-atom transfer radical addition reactions using 456 nm blue light. We further report protocols for the elaboration of these products to other useful derivatives, via iron-catalyzed Kumada coupling with aryl Grignard reagents and conversion of a pivalate ester to a carboxylic acid through hydrolysis/oxidation. The total times required to synthesize [3.1.1]propellane, the BCHep iodides and the BCHep carboxylic acid are ~53, 6-8 and 40 h, respectively, requiring an average level of synthetic chemistry expertise (for example, masters and/or graduate students).</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143441539","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":"Sonication-assisted protein extraction improves proteomic detection of membrane-bound and DNA-binding proteins from tumor tissues.","authors":"Qing Kay Li, T Mamie Lih, David J Clark, Lijun Chen, Michael Schnaubelt, Hui Zhang","doi":"10.1038/s41596-024-01113-9","DOIUrl":"10.1038/s41596-024-01113-9","url":null,"abstract":"<p><p>Deep-scale, mass spectrometry-based proteomic studies by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) program involves tissue lysis using urea buffer before data acquisition via mass spectrometry for quantitative global proteomic and phosphoproteomic analysis. This is described in a 2018 protocol¹. Here we report an update to this initial protocol by implementing a sonication step into urea-based tissue lysis. Similar to the initial CPTAC protocol, we identified >12,000 proteins and >25,000 phosphopeptides in a tandem mass tag (TMT) set containing both nonsonicated and sonicated tumor tissues from patient-derived xenograft mouse models. An improvement in the detection of membrane-bound and DNA-binding proteins was observed by including the sonication. We also offer recommendations for optimal sonication conditions such as the buffer composition, timing of sonication cycle, instrumentation settings and a troubleshooting section for potential users. Additionally, the protocol is equally applicable to other biological specimens.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143441514","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 connectome construction using constrained spherical deconvolution in multi-shell diffusion-weighted magnetic resonance imaging.","authors":"Marlene Tahedl, J-Donald Tournier, Robert E Smith","doi":"10.1038/s41596-024-01129-1","DOIUrl":"https://doi.org/10.1038/s41596-024-01129-1","url":null,"abstract":"<p><p>Connectional neuroanatomical maps can be generated in vivo by using diffusion-weighted magnetic resonance imaging (dMRI) data, and their representation as structural connectome (SC) atlases adopts network-based brain analysis methods. We explain the generation of high-quality SCs of brain connectivity by using recent advances for reconstructing long-range white matter connections such as local fiber orientation estimation on multi-shell dMRI data with constrained spherical deconvolution, which yields both increased sensitivity to detecting crossing fibers compared with competing methods and the ability to separate signal contributions from different macroscopic tissues, and improvements to streamline tractography such as anatomically constrained tractography and spherical-deconvolution informed filtering of tractograms, which have increased the biological accuracy of SC creation. Here, we provide step-by-step instructions to creating SCs by using these methods. In addition, intermediate steps of our procedure can be adapted for related analyses, including region of interest-based tractography and quantification of local white matter properties. The associated software MRtrix3 implements the relevant tools for easy application of the protocol, with specific processing tasks deferred to components of the FSL software. The protocol is suitable for users with expertise in dMRI and neuroscience and requires between 2 h and 13 h to complete, depending on the available computational system.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425744","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":"Single-cell CRISPR screening in mouse brain.","authors":"Eugenia V Pankevich, Christoph Bock","doi":"10.1038/s41596-024-01128-2","DOIUrl":"https://doi.org/10.1038/s41596-024-01128-2","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143409138","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":"Massively parallel in vivo Perturb-seq screening.","authors":"Xinhe Zheng, Patrick C Thompson, Cassandra M White, Xin Jin","doi":"10.1038/s41596-024-01119-3","DOIUrl":"https://doi.org/10.1038/s41596-024-01119-3","url":null,"abstract":"<p><p>Advances in genomics have identified thousands of risk genes impacting human health and diseases, but the functions of these genes and their mechanistic contribution to disease are often unclear. Moving beyond identification to actionable biological pathways requires dissecting risk gene function and cell type-specific action in intact tissues. This gap can in part be addressed by in vivo Perturb-seq, a method that combines state-of-the-art gene editing tools for programmable perturbation of genes with high-content, high-resolution single-cell genomic assays as phenotypic readouts. Here we describe a detailed protocol to perform massively parallel in vivo Perturb-seq using several versatile adeno-associated virus (AAV) vectors and provide guidance for conducting successful downstream analyses. Expertise in mouse work, AAV production and single-cell genomics is required. We discuss key parameters for designing in vivo Perturb-seq experiments across diverse biological questions and contexts. We further detail the step-by-step procedure, from designing a perturbation library to producing and administering AAV, highlighting where quality control checks can offer critical go-no-go points for this time- and cost-expensive method. Finally, we discuss data analysis options and available software. In vivo Perturb-seq has the potential to greatly accelerate functional genomics studies in mammalian systems, and this protocol will help others adopt it to answer a broad array of biological questions. From guide RNA design to tissue collection and data collection, this protocol is expected to take 9-15 weeks to complete, followed by data analysis.</p>","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-02-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143409116","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}