Nature Protocols最新文献_第2页

Advancing cell-cell communication analysis from single-cell genomics data.

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-25 DOI: 10.1038/s41596-025-01140-0

Iguaracy Pinheiro-de-Sousa, Evangelia Petsalaki

引用次数: 0

Gene expression and chromatin state mapped in space.

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-21 DOI: 10.1038/s41596-025-01151-x

Enric Llorens-Bobadilla

引用次数: 0

Spatially resolved genome-wide joint profiling of epigenome and transcriptome with spatial-ATAC-RNA-seq and spatial-CUT&Tag-RNA-seq.

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-21 DOI: 10.1038/s41596-025-01145-9

Haikuo Li, Shuozhen Bao, Negin Farzad, Xiaoyu Qin, Anthony A Fung, Di Zhang, Zhiliang Bai, Bo Tao, Rong Fan

{"title":"Spatially resolved genome-wide joint profiling of epigenome and transcriptome with spatial-ATAC-RNA-seq and spatial-CUT&Tag-RNA-seq.","authors":"Haikuo Li, Shuozhen Bao, Negin Farzad, Xiaoyu Qin, Anthony A Fung, Di Zhang, Zhiliang Bai, Bo Tao, Rong Fan","doi":"10.1038/s41596-025-01145-9","DOIUrl":"10.1038/s41596-025-01145-9","url":null,"abstract":"The epigenome of a cell is tightly correlated with gene transcription, which controls cell identity and diverse biological activities. Recent advances in spatial technologies have improved our understanding of tissue heterogeneity by analyzing transcriptomics or epigenomics with spatial information preserved, but have been mainly restricted to one molecular layer at a time. Here we present procedures for two spatially resolved sequencing methods, spatial-ATAC-RNA-seq and spatial-CUT&Tag-RNA-seq, that co-profile transcriptome and epigenome genome wide. In both methods, transcriptomic readouts are generated through tissue fixation, permeabilization and in situ reverse transcription. In spatial-ATAC-RNA-seq, Tn5 transposase is used to probe accessible chromatin, and in spatial-CUT&Tag-RNA-seq, the tissue is incubated with primary antibodies that target histone modifications, followed by Protein A-fused Tn5-induced tagmentation. Both methods leverage a microfluidic device that delivers two sets of oligonucleotide barcodes to generate a two-dimensional mosaic of tissue pixels at near single-cell resolution. A spatial-ATAC-RNA-seq or spatial-CUT&Tag-RNA-seq library can be generated in 3-5 d, allowing researchers to simultaneously investigate the transcriptomic landscape and epigenomic landscape of an intact tissue section. This protocol is an extension of our previous spatially resolved epigenome sequencing protocol and provides opportunities in multimodal profiling.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143677154","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

DARLIN mouse for in vivo lineage tracing at high efficiency and clonal diversity.

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-21 DOI: 10.1038/s41596-025-01141-z

Li Li, Sarah Bowling, Hongying Lin, Daolong Chen, Shou-Wen Wang, Fernando D Camargo

{"title":"DARLIN mouse for in vivo lineage tracing at high efficiency and clonal diversity.","authors":"Li Li, Sarah Bowling, Hongying Lin, Daolong Chen, Shou-Wen Wang, Fernando D Camargo","doi":"10.1038/s41596-025-01141-z","DOIUrl":"https://doi.org/10.1038/s41596-025-01141-z","url":null,"abstract":"Lineage tracing is a powerful tool to study cell history and cell dynamics during tissue development and homeostasis. An increasingly popular approach for lineage tracing is to generate high-frequent mutations at given genomic loci, which can serve as genetic barcodes to label different cell lineages. However, current lineage tracing mouse models suffer from low barcode diversity and limited single-cell lineage coverage. We recently developed the DARLIN mouse model by incorporating three barcoding arrays within defined genomic loci and combining Cas9 and terminal deoxynucleotidyl transferase (TdT) to improve editing diversity in each barcode array. We estimated that DARLIN generates 1018 distinct lineage barcodes in theory, and enables the recovery of lineage barcodes in over 70% of cells in single-cell assays. In addition, DARLIN can be induced with doxycycline to generate stable lineage barcodes across different tissues at a defined stage. Here we provide a step-by-step protocol on applying the DARLIN system for in vivo lineage tracing, including barcode induction, estimation of induction efficiency, barcode analysis with bulk and single-cell sequencing, and computational analysis. The execution time of this protocol is ~1 week for experimental data collection and ~1 d for running the computational analysis pipeline. To execute this protocol, one should be familiar with sequencing library generation and Linux operation. DARLIN opens the door to study the lineage relationships and the underlying molecular regulations across various tissues at physiological context.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143677150","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

Standardized measurements for monitoring and comparing multiphoton microscope systems.

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-17 DOI: 10.1038/s41596-024-01120-w

Robert M Lees, Isaac H Bianco, Robert A A Campbell, Natalia Orlova, Darcy S Peterka, Bruno Pichler, Spencer LaVere Smith, Dimitri Yatsenko, Che-Hang Yu, Adam M Packer

引用次数: 0

Live-cell synthesis of biocompatible quantum dots.

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-17 DOI: 10.1038/s41596-024-01133-5

An-An Liu, Ran Cui, Xia Zong, Jianhong Jia, Yusi Hu, Jing-Ya Zhao, Dai-Wen Pang

{"title":"Live-cell synthesis of biocompatible quantum dots.","authors":"An-An Liu, Ran Cui, Xia Zong, Jianhong Jia, Yusi Hu, Jing-Ya Zhao, Dai-Wen Pang","doi":"10.1038/s41596-024-01133-5","DOIUrl":"https://doi.org/10.1038/s41596-024-01133-5","url":null,"abstract":"Quantum dots (QDs) exhibit fluorescence properties with promising prospects for biomedical applications; however, the QDs synthesized in organic solvents shows poor biocompatibility, limiting their use in biological systems. We developed an approach for synthesizing QDs in live cells by coupling a series of intracellular metabolic pathways in a precise spatial and temporal sequence. We have validated this approach in yeast (Saccharomyces cerevisiae), Staphylococcus aureus, Michigan Cancer Foundation-7 (MCF-7) and Madin-Darby canine kidney (MDCK) cells. The intracellularly synthesized QDs are inherently stable and biocompatible, making them suitable for the direct in situ labeling of cells and cell-derived vesicles. Here, we provide an optimized workflow for the live-cell synthesis of QDs by using S. cerevisiae, S. aureus or MCF-7 cells. In addition, we detail a cell-free aqueous synthetic system (quasi-biosynthesis) containing enzymes, electrolytes, peptides and coenzymes, which closely mimics the intracellular synthetic conditions used in our cell culture system. In this solution, we synthesize biocompatible ultrasmall QDs that are easier to purify and characterize than those synthesized in cells. The live-cell-synthesized QDs can be used for bioimaging and microvesicle detection, whereas the quasi-biosynthesized QDs are suitable for applications such as biodetection, biolabeling and real-time imaging. The procedure can be completed in 3-4 d for live-cell QD synthesis and 2 h for the quasi-biosynthesis of QDs. The procedure is suitable for users with expertise in chemistry, biology, materials science and synthetic biology. This approach encourages interested researchers to engage in the field of QDs and develop further biomedical applications.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143649761","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

Live STED imaging of functional neuroanatomy. 功能神经解剖学的实时 STED 成像。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-14 DOI: 10.1038/s41596-024-01132-6

Misa Arizono, Agata Idziak, U Valentin Nägerl

{"title":"Live STED imaging of functional neuroanatomy.","authors":"Misa Arizono, Agata Idziak, U Valentin Nägerl","doi":"10.1038/s41596-024-01132-6","DOIUrl":"https://doi.org/10.1038/s41596-024-01132-6","url":null,"abstract":"In the mammalian brain, a large network of excitable and modulatory cells efficiently processes, analyzes and stores vast amounts of information. The brain's anatomy influences the flow of neural information between neurons and glia, from which all thought, emotion and action arises. Consequently, one of the grand challenges in neuroscience is to uncover the finest structural details of the brain in the context of its overall architecture. Recent developments in microscopy and biosensors have enabled the investigation of brain microstructure and function with unprecedented specificity and resolution, dendritic spines being an exemplary case, which has provided deep insights into neuronal mechanisms of higher brain function, such as learning and memory. As diffraction-limited light microscopy methods cannot resolve the fine details of brain cells (the 'anatomical ground truth'), electron microscopy is used instead to contextualize functional signals. This approach can be quite unsatisfying given the fragility and dynamic nature of the structures under investigation. We have recently developed a method for combining super-resolution stimulated emission depletion microscopy with functional measurements in brain slices, offering nanoscale resolution in functioning brain structures. We describe how to concurrently perform morphological and functional imaging with a confocal STED microscope. Specifically, the procedure guides the user on how to record astrocytic Ca2+ signals at tripartite synapses, outlining a framework for analyzing structure-function relationships of brain cells at nanoscale resolution. The imaging requires 2-3 h and the image analysis between 2 h and 2 d.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143634232","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

Preparation, maintenance and propagation of synchronous cultures of photoactive Chlamydomonas cells.

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-13 DOI: 10.1038/s41596-024-01135-3

Rodrigo E Catalan, Alexandros A Fragkopoulos, Antoine Girot, Maike Lorenz, Oliver Bäumchen

{"title":"Preparation, maintenance and propagation of synchronous cultures of photoactive Chlamydomonas cells.","authors":"Rodrigo E Catalan, Alexandros A Fragkopoulos, Antoine Girot, Maike Lorenz, Oliver Bäumchen","doi":"10.1038/s41596-024-01135-3","DOIUrl":"https://doi.org/10.1038/s41596-024-01135-3","url":null,"abstract":"The systematic cultivation of species of photosynthetically active 'green' microorganisms in research labs started in the 1940s. Among these microorganisms, Chlamydomonas represents a genus of green biciliated microalgae, of which Chlamydomonas reinhardtii has become the main describing species. For decades C. reinhardtii has been used as an established model organism in biology, including research areas such as molecular biology of eukaryotes, photosynthesis, light receptors, cell metabolism, the dynamics of microtubule assembly and protein transport along cilia. More recently, the use of suspensions of light-responsive living microorganisms has seen a major expansion from the life sciences to the biophysics, statistical physics, fluid dynamics and bioengineering communities. Studies that substantially advance the state of the art in these research areas require the reliable preparation and maintenance of viable, monodisperse and synchronous cell cultures. Although some technical aspects are shared with standard procedures in cell biology and microbiology, Chlamydomonas and its relatives are photosensitive and, simultaneously, motile, meaning this microorganism requires tailored cultivation protocols that are specific to this species. Here we provide guidance on which Chlamydomonas wild-type and mutant strains are suitable for specific experiments and provide detailed step-by-step procedures to measure culture synchronicity, growth rate of the population, average cell size and motility features. The reliable preparation of cell cultures may facilitate future interdisciplinary research using living suspensions of photoactive microorganisms.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143625306","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 translatable IR-chemometrics model for the rapid prediction of structural and material properties of technical lignins.

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-12 DOI: 10.1038/s41596-025-01139-7

Luke A Riddell, Peter de Peinder, Jean-Pierre B Lindner, Florian Meirer, Pieter C A Bruijnincx

{"title":"A translatable IR-chemometrics model for the rapid prediction of structural and material properties of technical lignins.","authors":"Luke A Riddell, Peter de Peinder, Jean-Pierre B Lindner, Florian Meirer, Pieter C A Bruijnincx","doi":"10.1038/s41596-025-01139-7","DOIUrl":"https://doi.org/10.1038/s41596-025-01139-7","url":null,"abstract":"Technical lignins are an industrial byproduct of plant biomass processing, for example, paper production or biorefinery operations. They are highly functional and aromatic, making them potentially suitable for a diverse range of applications; however, their exact structural composition depends on the plant species and the industrial process involved. A major bottleneck to lignin valorization and to biorefining in general is the equipment and time investment required for the full characterization of each sample. An array of wet chemical, spectroscopic, chromatographic and thermal methods are typically required to effectively characterize a given lignin sample. To ease the analytical burden, measured lignin properties can be correlated with detailed spectroscopic data obtained from a rapid analytical technique, such as attenuated total reflectance (ATR) Fourier-transform infrared (IR) spectroscopy, which requires minimal sample preparation. With sufficient sensitivity of the spectroscopic data, partial least squares regression models can be calibrated and, thus, predict these properties for future samples for which only the ATR-IR spectra are recorded. So far, several structural and macromolecular properties of lignin have been correlated with ATR-IR spectral data and quantitatively predicted in such a manner, including molecular weight, hydroxyl group content ([OH]), interunit linkage abundance and glass transition temperature. The protocol to apply this powerful lignin characterization methodology is described herein. Here, we also present a simple calibration transfer step, which when implemented before partial least squares regression, addresses the problem of instrument dependency. With the calibrated model, it is possible to determine lignin properties from a single ATR-IR spectral measurement (in ~5 min per sample).","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143615494","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

mRNA lipid nanoparticle formulation, characterization and evaluation.

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-03-11 DOI: 10.1038/s41596-024-01134-4

Yutian Ma, Rachel VanKeulen-Miller, Owen S Fenton

{"title":"mRNA lipid nanoparticle formulation, characterization and evaluation.","authors":"Yutian Ma, Rachel VanKeulen-Miller, Owen S Fenton","doi":"10.1038/s41596-024-01134-4","DOIUrl":"https://doi.org/10.1038/s41596-024-01134-4","url":null,"abstract":"mRNA-based therapies have emerged as a cutting-edge approach for diverse therapeutic applications. However, substantial barriers exist that hinder scientists from entering this research field, including the technical complexity and multiple potential workflows available for formulating and evaluating mRNA lipid nanoparticles (LNPs). Here we present an easy-to-follow and step-by-step guide for mRNA LNP formulation, characterization and in vitro and in vivo evaluation that could lower these barriers, facilitating entry for scientists in academia, industry and clinical settings into this research space. In this protocol, we detail steps for formulating representative mRNA LNPs (0.5 d) and characterizing key parameters (1-6 d) such as size, polydispersity index, zeta potential, mRNA concentration, mRNA encapsulation efficiency and stability. Then, we describe in vitro evaluations (3-6 d), such as protein expression, cell uptake and mechanism investigations (3-5 d), including endosomal escape, as well as in vivo delivery evaluation (2-3 d) encompassing intracellular and secreted protein expression levels, biodistribution and additional tolerability studies (1-2 weeks). Unlike some alternative protocols that may focus on discrete aspects of the workflow-such as formulation, characterization or evaluation-our protocol instead aims to integrate each of these aspects into a simplified, singular workflow applicable across multiple types of mRNA LNP formulations. In describing these procedures, we wish to disseminate one potential workflow for mRNA LNP production and evaluation, with the ultimate goal of furthering innovation, collaboration and the translational advancement of mRNA LNPs.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143605640","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