Nature Protocols最新文献

Author Correction: Transcriptomic profiling of individual bacteria by MATQ-seq. 作者更正：通过MATQ-seq对单个细菌进行转录组学分析。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-30 DOI: 10.1038/s41596-025-01213-0

Christina Homberger, Fabian Imdahl, Regan J Hayward, Lars Barquist, Antoine-Emmanuel Saliba, Jörg Vogel

引用次数: 0

Fibrous polyisocyanide hydrogels for 3D cell culture applications. 纤维多异氰化物水凝胶用于3D细胞培养应用。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-30 DOI: 10.1038/s41596-025-01159-3

Hongbo Yuan, Kaizheng Liu, Melissa J J van Velthoven, Jyoti Kumari, Yuying Bao, Susana Rocha, Paul H J Kouwer

{"title":"Fibrous polyisocyanide hydrogels for 3D cell culture applications.","authors":"Hongbo Yuan, Kaizheng Liu, Melissa J J van Velthoven, Jyoti Kumari, Yuying Bao, Susana Rocha, Paul H J Kouwer","doi":"10.1038/s41596-025-01159-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01159-3","url":null,"abstract":"Three-dimensional (3D) cell culture models based on hydrogels are rapidly evolving into a prominent tool for tissue engineering, mechanobiology, disease modeling and drug screening. While a vast variety of synthetic gels have emerged in recent years, they fail to penetrate the market substantially for two major reasons: they poorly mimic the extracellular matrix or they are difficult to use in gel formation and cell extraction. Mimicking the complexity of nature is challenging: the extracellular matrix plays a crucial role in cell development and function, which goes well beyond simple mechanical support. Recently, we introduced polyisocyanide (PIC) hydrogels for 3D cell culture applications. The fibrous architecture and associated (non)linear mechanical behavior closely mimic the physical properties of biogels such as collagen and fibrin. As fully synthetic materials, PIC gels benefit from high tailorability and reproducibility. Moreover, the thermoresponsive properties of PIC gels make them easy to handle in the lab; the gels form instantly at 37 °C and cells are easily extracted after cooling to 5 °C. The potential of PIC gels has been demonstrated in a quickly expanding library of papers discussing different cell lines, primary cells and organoids, as well as in vivo experiments. This manuscript provides protocols on how to handle PIC gels in the chemistry and cell biology laboratories. Material preparation requires 72 h. Cell encapsulation takes 1 h and the time for downstream analysis depends on the (commercial) methods used. The protocols described are suitable for researchers with expertise in cell culture and molecular biology.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144187418","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 scalable protocol for the radiosynthesis of clinical grade lutetium-177-labeled theranostic agents. 放射合成临床级镥-177标记治疗剂的可扩展方案。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-27 DOI: 10.1038/s41596-025-01176-2

William W Hunt, Mathew Long, Usama Kamil, Sunil Kellapatha, Wayne Noonan, Peter D Roselt, Brittany Emmerson, Michael S Hofman, Mohammad B Haskali

{"title":"A scalable protocol for the radiosynthesis of clinical grade lutetium-177-labeled theranostic agents.","authors":"William W Hunt, Mathew Long, Usama Kamil, Sunil Kellapatha, Wayne Noonan, Peter D Roselt, Brittany Emmerson, Michael S Hofman, Mohammad B Haskali","doi":"10.1038/s41596-025-01176-2","DOIUrl":"https://doi.org/10.1038/s41596-025-01176-2","url":null,"abstract":"Theranostics utilizes tandem targeted diagnostic and therapeutic agents that are molecularly analogous. In a theranostic approach, the diagnostic agent is a tracer typically radiolabeled with a positron emission tomography radionuclide such as fluorine-18 or gallium-68. Utilizing the selectivity of the tracer, the therapeutic agent is subsequently radiolabeled with an ablative radionuclide such as the β- emitting lanthanide lutetium-177 (177Lu). 177Lu is typically incorporated into theranostics using the chelators 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetraacetic acid (DOTA) and 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA) that are used to prepare the 177Lu-radiopharmaceutical [177Lu]Lu-DOTA-TATE, [177Lu]Lu-PSMA-617 and [177Lu]Lu-PSMA-I&T. Here we describe the scalable and validated production for these 177Lu-radiopharmaceuticals and further include the necessary quality control protocols. The procedures can be generalized and support both carrier added and noncarrier added 177Lu sources for use in a clinical setting. With robust procedures that accommodate 177Lu activity levels from 5 to 100 GBq, the procedures ensure stability for up to 8 h postproduction and achieve an average activity yield of 98%. As proven in over 1,000 patient cycles, this methodology is adaptable to both centralized production facilities and regional centers, enabling versatile application across small and large-scale production settings.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144159740","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

Hybridization chain reaction-based DNA nanoframeworks for biosensing and therapeutic applications. 基于杂交链反应的DNA纳米框架的生物传感和治疗应用。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-26 DOI: 10.1038/s41596-025-01183-3

Zhaoyue Lv, Peiran Li, Mingxing Liu, Chi Yao, Dayong Yang

{"title":"Hybridization chain reaction-based DNA nanoframeworks for biosensing and therapeutic applications.","authors":"Zhaoyue Lv, Peiran Li, Mingxing Liu, Chi Yao, Dayong Yang","doi":"10.1038/s41596-025-01183-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01183-3","url":null,"abstract":"Artificial DNA nanostructures, with their sequence programmability, precise molecular recognition and tunable stimuli responsiveness, bridge material chemistry and biomedicine. Here we detail the design and construction of hybridization chain reaction (HCR)-based DNA nanoframeworks, a class of DNA nanostructures with programmable sequences and customizable functions. HCR is an efficient, enzyme-free amplification strategy that isothermally produces nicked double-stranded DNA with periodically repeated modules via the assembly of two DNA hairpins, triggered by a DNA initiator. In contrast to other available assembly methods for the synthesis of DNA nanostructures, such as tile-mediated assembly, DNA origami and rolling circle amplification, the HCR method offers improved stability and efficiency under mild conditions, without reliance on enzymatic activity. The procedure uses radical polymerization to integrate DNA initiator into nanoframeworks, with overhangs complementary to functional sequences - termed linkers -which are amplified and incorporated through HCR. The linkers enable the incorporation of functional nucleic acid sequences. The HCR-based DNA nanoframeworks facilitate the loading capability of the delivered molecules, showing notable therapeutic efficacy and biosensing sensitivity. Preparation time for HCR-based DNA nanoframeworks ranges from 30 h to 45 h, depending on the payload.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144151147","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

Facile synthesis of mesoporous TiO₂ architectures with tunable configurations and nanometer precision. 具有可调结构和纳米精度的介孔TiO2结构的简单合成。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-23 DOI: 10.1038/s41596-025-01175-3

Jingyu Zhang, Jialong Li, Xu Wen, Lu Liu, Qiulong Wei, Pengfei Zhang, Jun-Ye Zhang, Dongyuan Zhao, Kun Lan

{"title":"Facile synthesis of mesoporous TiO2 architectures with tunable configurations and nanometer precision.","authors":"Jingyu Zhang, Jialong Li, Xu Wen, Lu Liu, Qiulong Wei, Pengfei Zhang, Jun-Ye Zhang, Dongyuan Zhao, Kun Lan","doi":"10.1038/s41596-025-01175-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01175-3","url":null,"abstract":"Titanium dioxide (TiO2) can be used in various applications such as catalysis, sensing, energy storage and conversion due to its semiconducting and crystalline properties. Ordered mesoporous TiO2 materials with high porosity values may further enable improvements in mass diffusion and surface access. However, despite the syntheses of TiO2-based bulks and polymorphs in the past, it remains challenging to control mesoscopic TiO2 morphology and dimension, pore shape and size, crystallographic phase and orientation. Here we describe a facile and robust solution-processed methodology for the preparation of a series of mesoporous TiO2 structures with highly tailored architectures at the atomic scale, nanoscale and mesoscale. The process relies on the preformation of flexible micelle hydrogels and the stepwise assembly, under specific conditions to synthesize diverse mesoporous TiO2 configurations with tunable parameters, such as bouquet-like spheres, chapped spheres, monolayered nanosheets, sandwich, vertical films and so on. The synthetic conditions and procedures are provided in detail to ensure the reproducibility of the experiments. The preparation of micelle hydrogels takes ~21 h, and the subsequent synthesis time to obtain versatile mesoporous TiO2 is usually ~50 h. Our protocol is suitable for researchers in nanomaterials, porous and inorganic materials and other related disciplines.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144132686","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

Integrating optical neuroscience tools into touchscreen operant systems. 将光学神经科学工具集成到触摸屏操作系统中。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-23 DOI: 10.1038/s41596-025-01143-x

Patrick T Piantadosi, Oren Princz-Lebel, Miguel Skirzewski, Julie R Dumont, Daniel Palmer, Sara Memar, Lisa M Saksida, Vania F Prado, Marco A M Prado, Tim J Bussey, Andrew Holmes

{"title":"Integrating optical neuroscience tools into touchscreen operant systems.","authors":"Patrick T Piantadosi, Oren Princz-Lebel, Miguel Skirzewski, Julie R Dumont, Daniel Palmer, Sara Memar, Lisa M Saksida, Vania F Prado, Marco A M Prado, Tim J Bussey, Andrew Holmes","doi":"10.1038/s41596-025-01143-x","DOIUrl":"https://doi.org/10.1038/s41596-025-01143-x","url":null,"abstract":"Unlocking the neural regulation of complex behavior is a foundational goal of brain science. Touchscreen-based assessments of behavior have been used extensively in the pursuit of this goal, with traditional pharmacological and neurochemical approaches being employed to provide key insights into underlying neural systems. So far, optically based approaches to measure and manipulate neural function, which have begun to revolutionize our understanding of relatively simple behaviors, have been less widely adopted for more complex cognitive functions of the type assessed with touchscreen-based behavioral tasks. Here we provide guidance and procedural descriptions to enable researchers to integrate optically based manipulation and measurement techniques into their touchscreen experimental systems. We focus primarily on three techniques, optogenetic manipulation, fiber photometry and microendoscopic imaging, describing experimental design adjustments that we have found to be critical to the successful integration of these approaches with extant touchscreen behavior pipelines. These include factors related to surgical procedures and timing, alterations to touchscreen operant environments and approaches to synchronizing light delivery and task design. A detailed protocol is included for each of the three techniques, covering their use from implementation through data analysis. The procedures in this protocol can be conducted in as short a time as a few days or over the course of weeks or months.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144132688","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

Solid-phase DNA-encoded library synthesis: a master builder's instructions. 固相dna编码文库合成：建造大师的指令。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-22 DOI: 10.1038/s41596-025-01190-4

Anjali Dixit, Brian M Paegel

{"title":"Solid-phase DNA-encoded library synthesis: a master builder's instructions.","authors":"Anjali Dixit, Brian M Paegel","doi":"10.1038/s41596-025-01190-4","DOIUrl":"https://doi.org/10.1038/s41596-025-01190-4","url":null,"abstract":"Solid-phase DNA-encoded library (DEL) synthesis is a next-generation drug discovery technology with powerful activity-based and cellular lead identification capabilities. Solid-phase DELs combine the one-bead-one-compound approach with DNA encoding to furnish beads that display multiple copies of photocleavable library members and DNA encoding tags. Sequential chemical synthesis and enzymatic DNA ligation reactions yield an encoded library in which individual library members are physically isolable, enabling various high-throughput screening modalities. This advancement from on-DNA synthesis, in which small molecules are directly attached to their DNA-encoding tags, decouples the library member from the steric bulk of the DNA tag, which prevents biased binding to a target. Here we provide step-by-step instructions for solid-phase DEL synthesis, incorporating all of our most recent quality control innovations to ensure robust library production. The protocol begins with on-bead synthesis of a linker containing a spectroscopic handle for chromatographic analysis, an ionization enhancer for mass spectrometry and an alkyne for installation of DNA encoding sites via copper-catalyzed azide-alkyne cycloaddition click chemistry. Coupling of a photocleavable linker before library synthesis enables compound liberation from the bead for activity-based screening. Powerful combinatorial split-and-pool parallel synthesis tactics transform modest collections of small-molecule building blocks into large DELs of all possible building block combinations. Post synthesis, decoding and mass analysis of single DEL beads as well as whole-library deep sequencing provides rigorous chemical and bioinformatic quality control and establishes suitability for screening. The solid-phase chemistry is highly accessible: expertise in chemical synthesis is not necessary and solid-phase synthesis apparatus is routinely available in molecular biology laboratories. This procedure requires ~1 month to complete.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128209","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

Single microorganism RNA sequencing of microbiomes using smRandom-Seq. 使用smRandom-Seq对微生物组进行单微生物RNA测序。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-22 DOI: 10.1038/s41596-025-01181-5

Ziye Xu, Yuting Wang, Wenjie Cai, Yu Chen, Yongcheng Wang

{"title":"Single microorganism RNA sequencing of microbiomes using smRandom-Seq.","authors":"Ziye Xu, Yuting Wang, Wenjie Cai, Yu Chen, Yongcheng Wang","doi":"10.1038/s41596-025-01181-5","DOIUrl":"https://doi.org/10.1038/s41596-025-01181-5","url":null,"abstract":"Bacteria colonize nearly every part of the human body and various environments, displaying remarkable diversity. Traditional population-level transcriptomics measurements provide only average population behaviors, often overlooking the heterogeneity within bacterial communities. To address this limitation, we have developed a droplet-based, high-throughput single-microorganism RNA sequencing method (smRandom-seq) that offers highly species specific and sensitive gene detection. Here we detail procedures for microbial sample preprocessing, in situ preindexed cDNA synthesis, in situ poly(dA) tailing, droplet barcoding, ribosomal RNA depletion and library preparation. The main smRandom-seq workflow, including sample processing, in situ reactions and library construction, takes ~2 days. This method features enhanced RNA coverage, reduced doublet rates and minimized ribosomal RNA contamination, thus enabling in-depth analysis of microbial heterogeneity. smRandom-seq is compatible with microorganisms from both laboratory cultures and complex microbial community samples, making it well suited for constructing single-microorganism transcriptomic atlases of bacterial strains and diverse microbial communities. This Protocol requires experience in molecular biology and RNA sequencing techniques, and it holds promising potential for researchers investigating bacterial resistance, microbiome heterogeneity and host-microorganism interactions.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144128206","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

Integration of diverse bioactivity data into the Chemical Checker compound universe. 将多种生物活性数据整合到化学检查器化合物宇宙中。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-21 DOI: 10.1038/s41596-025-01167-3

Arnau Comajuncosa-Creus, Martino Bertoni, Miquel Duran-Frigola, Adrià Fernández-Torras, Oriol Guitart-Pla, Nils Kurzawa, Martina Locatelli, Yasmmin Martins, Elena Pareja-Lorente, Gema Rojas-Granado, Nicolas Soler, Eva Viesi, Patrick Aloy

{"title":"Integration of diverse bioactivity data into the Chemical Checker compound universe.","authors":"Arnau Comajuncosa-Creus, Martino Bertoni, Miquel Duran-Frigola, Adrià Fernández-Torras, Oriol Guitart-Pla, Nils Kurzawa, Martina Locatelli, Yasmmin Martins, Elena Pareja-Lorente, Gema Rojas-Granado, Nicolas Soler, Eva Viesi, Patrick Aloy","doi":"10.1038/s41596-025-01167-3","DOIUrl":"https://doi.org/10.1038/s41596-025-01167-3","url":null,"abstract":"Chemical signatures encode the physicochemical and structural properties of small molecules into numerical descriptors, forming the basis for chemical comparisons and search algorithms. The increasing availability of bioactivity data has improved compound representations to include biological effects (for example, induced gene expression changes), although bioactivity descriptors are often limited to a few well-documented molecules. To address this issue, we implemented a collection of deep neural networks able to leverage the experimentally determined bioactivity data associated to small molecules and infer the missing bioactivity signatures for any compound of interest. However, unlike static chemical descriptors, these bioactivity signatures dynamically evolve with new data and processing strategies. Here we present a computational protocol to modify or generate novel bioactivity spaces and signatures, describing the main steps needed to leverage diverse bioactivity data with the current knowledge, as catalogued in the Chemical Checker (CC; https://chemicalchecker.org/ ), using the predefined data curation pipeline. We illustrate the functioning of the protocol through four specific examples, including the incorporation of new compounds to an already existing bioactivity space, a change in the data preprocessing without altering the underlying experimental data and the creation of two novel bioactivity spaces from scratch, which are completed in under 9 h using graphics processing unit computing. Overall, this protocol offers a guideline for installing, testing and running the CC data integration approach on user-provided data, extending the annotation presented for a limited number of small molecules to a larger chemical landscape and generating novel bioactivity signatures.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144120344","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

Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR. 用16S rRNA qPCR或ddPCR对微生物组中的原核生物进行绝对定量。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2025-05-19 DOI: 10.1038/s41596-025-01165-5

Boryana Doyle, Gabriella Z M Reynolds, Mai Dvorak, Dylan G Maghini, Aravind Natarajan, Ami S Bhatt

{"title":"Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR.","authors":"Boryana Doyle, Gabriella Z M Reynolds, Mai Dvorak, Dylan G Maghini, Aravind Natarajan, Ami S Bhatt","doi":"10.1038/s41596-025-01165-5","DOIUrl":"https://doi.org/10.1038/s41596-025-01165-5","url":null,"abstract":"Measurements of prokaryotic absolute abundance can provide important insights into human gut microbiome biology and correct misinterpretations of relative abundance data. Despite the existence of several relatively well-established methods for making these measurements, most microbiome studies do not report absolute abundance. To enable researchers equipped with standard molecular biology capabilities to incorporate absolute quantification into their microbiome studies, we present a detailed, step-by-step protocol for rigorous and reproducible quantification of prokaryotic concentration in stool samples. We include methods for measuring stool sample moisture content, quantifying the concentration of the 16S rRNA prokaryotic marker gene by qPCR or digital droplet PCR (ddPCR) and analyzing the resulting data. We also highlight and provide strategies to overcome common pitfalls of the quantification method, such as 16S rRNA gene contamination. The final output of this approach is 16S rRNA copies per wet or dry gram of stool. In cases where samples have matched metagenomic sequencing information, data can be converted into absolute concentration of prokaryotes and taxon-specific absolute concentrations. To enable researchers to choose the appropriate method for their specific applications, we also compare and contrast our qPCR and ddPCR methods. In 4 days, ~80 samples can be taken from frozen stool to absolute concentration by using qPCR or ddPCR without the need for resequencing. Overall, this protocol provides a sensitive and straightforward way to measure the absolute concentration of prokaryotes in human gut microbiome samples stored with or without preservative.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2025-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144102220","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