Nature Protocols最新文献_第2页

Seq-Scope: repurposing Illumina sequencing flow cells for high-resolution spatial transcriptomics. Seq-Scope：重新利用 Illumina 测序流式细胞进行高分辨率空间转录组学研究。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-10-31 DOI: 10.1038/s41596-024-01065-0

Yongsung Kim, Weiqiu Cheng, Chun-Seok Cho, Yongha Hwang, Yichen Si, Anna Park, Mitchell Schrank, Jer-En Hsu, Angelo Anacleto, Jingyue Xi, Myungjin Kim, Ellen Pedersen, Olivia I Koues, Thomas Wilson, ChangHee Lee, Goo Jun, Hyun Min Kang, Jun Hee Lee

{"title":"Seq-Scope: repurposing Illumina sequencing flow cells for high-resolution spatial transcriptomics.","authors":"Yongsung Kim, Weiqiu Cheng, Chun-Seok Cho, Yongha Hwang, Yichen Si, Anna Park, Mitchell Schrank, Jer-En Hsu, Angelo Anacleto, Jingyue Xi, Myungjin Kim, Ellen Pedersen, Olivia I Koues, Thomas Wilson, ChangHee Lee, Goo Jun, Hyun Min Kang, Jun Hee Lee","doi":"10.1038/s41596-024-01065-0","DOIUrl":"10.1038/s41596-024-01065-0","url":null,"abstract":"Spatial transcriptomics technologies aim to advance gene expression studies by profiling the entire transcriptome with intact spatial information from a single histological slide. However, the application of spatial transcriptomics is limited by low resolution, limited transcript coverage, complex procedures, poor scalability and high costs of initial setup and/or individual experiments. Seq-Scope repurposes the Illumina sequencing platform for high-resolution, high-content spatial transcriptome analysis, overcoming these limitations. It offers submicrometer resolution, high capture efficiency, rapid turnaround time and precise annotation of histopathology at a much lower cost than commercial alternatives. This protocol details the implementation of Seq-Scope with an Illumina NovaSeq 6000 sequencing flow cell, allowing the profiling of multiple tissue sections in an area of 7 mm × 7 mm or larger. We describe the preparation of a fresh-frozen tissue section for both histological imaging and sequencing library preparation and provide a streamlined computational pipeline with comprehensive instructions to integrate histological and transcriptomic data for high-resolution spatial analysis. This includes the use of conventional software tools for single-cell and spatial analysis, as well as our recently developed segmentation-free method for analyzing spatial data at submicrometer resolution. Aside from array production and sequencing, which can be done in batches, tissue processing, library preparation and running the computational pipeline can be completed within 3 days by researchers with experience in molecular biology, histology and basic Unix skills. Given its adaptability across various biological tissues, Seq-Scope establishes itself as an invaluable tool for researchers in molecular biology and histology.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-10-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142558286","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

Outcome devaluation as a method for identifying goal-directed behaviors in rats. 用结果贬值法识别大鼠的目标导向行为

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-10-23 DOI: 10.1038/s41596-024-01054-3

Zachary Pierce-Messick, Megan L Shipman, Gabrielle L Desilets, Laura H Corbit

{"title":"Outcome devaluation as a method for identifying goal-directed behaviors in rats.","authors":"Zachary Pierce-Messick, Megan L Shipman, Gabrielle L Desilets, Laura H Corbit","doi":"10.1038/s41596-024-01054-3","DOIUrl":"https://doi.org/10.1038/s41596-024-01054-3","url":null,"abstract":"Goal-directed behaviors allow animals to act to satisfy needs and desires. The outcome devaluation task is an effective method for identifying goal-directed behaviors and distinguishing these from other types of behavior. Rats can be trained to lever-press for one or multiple distinct food rewards. During testing, the previously earned food-or a control food for comparison-is devalued by allowing the animal to freely feed on it until they are sated before testing lever-press performance under extinction conditions (no rewards are delivered). Behavior that adapts to reflect the new value of the outcome is considered goal-directed, whereas behavior that continues as in previous training despite the change in outcome value, is not. As more research groups have used this task, variability in the procedures used has increased. Here, we provide a reliable procedure for conducting the outcome devaluation task with appropriate controls. We describe the most common variants of the task and control conditions and discuss troubleshooting measures such as outcome pre-exposure, habituation to pre-feeding chambers and attention to animals' hunger levels. The method outlined can be executed in ~2 weeks including training (~8 d) and testing (1-4 d) by researchers who are familiar with performing behavioral tasks in laboratory rodents, although longer training may be considered for those who are interested in observing habitual control of behavior. This protocol should facilitate the comparison of results from different studies and laboratories, while allowing flexibility in the application of the outcome devaluation task to different research questions.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504448","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

High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array. 利用基于化学探针的阵列对细胞外囊泡进行高通量捕获和原位蛋白质分析。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-10-22 DOI: 10.1038/s41596-024-01082-z

Xin Feng, Ao Shen, Wei Zhang, Shengnan Jia, Anton Iliuk, Yuling Wang, Wenke Zhang, Ying Zhang, W Andy Tao, Lianghai Hu

{"title":"High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array.","authors":"Xin Feng, Ao Shen, Wei Zhang, Shengnan Jia, Anton Iliuk, Yuling Wang, Wenke Zhang, Ying Zhang, W Andy Tao, Lianghai Hu","doi":"10.1038/s41596-024-01082-z","DOIUrl":"https://doi.org/10.1038/s41596-024-01082-z","url":null,"abstract":"Extracellular vesicles (EVs) are small particles with phospholipid bilayers that carry a diverse range of cargoes including nucleic acids, proteins and metabolites. EVs have important roles in various cellular processes and are increasingly recognized for their ubiquitous role in cell-cell communications and potential applications in therapeutics and diagnostics. Although many methods have been developed for the characterization and measurement of EVs, analyzing them from biofluids remains a challenge with regard to throughput and sensitivity. Recently, we introduced an approach to facilitate high-throughput analysis of EVs from trace amounts of sample. In this method, an amphiphile-dendrimer supramolecular probe (ADSP) is coated onto a nitrocellulose membrane for array-based capture and to enable an in situ immunoblotting assay. Here, we describe the protocol for our array-based method of EV profiling. We describe an enhanced version of the method that incorporates an automated printing workstation, ensuring high throughput and reproducibility. We further demonstrate the use of our array to profile specific glycosylations on the EV surface using click chemistry of an azide group introduced by metabolic labeling. In this protocol, the synthesis of ADSP and the fabrication of ADSP nitrocellulose membrane array can be completed on the same day. EVs are efficiently captured from biological or clinical samples through a 30-min incubation, followed by an immunoblotting assay within a 3-h window, thus providing a high-throughput platform for EV isolation and in situ targeted analysis of EV proteins and their modifications.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504447","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

Validation and quantification of peptide antigens presented on MHCs using SureQuant. 使用 SureQuant 验证和量化呈现在 MHC 上的多肽抗原。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-10-22 DOI: 10.1038/s41596-024-01076-x

Owen Leddy, Yufei Cui, Ryuhjin Ahn, Lauren Stopfer, Elizabeth Choe, Do Hun Kim, Malte Roerden, Stefani Spranger, Bryan D Bryson, Forest M White

{"title":"Validation and quantification of peptide antigens presented on MHCs using SureQuant.","authors":"Owen Leddy, Yufei Cui, Ryuhjin Ahn, Lauren Stopfer, Elizabeth Choe, Do Hun Kim, Malte Roerden, Stefani Spranger, Bryan D Bryson, Forest M White","doi":"10.1038/s41596-024-01076-x","DOIUrl":"https://doi.org/10.1038/s41596-024-01076-x","url":null,"abstract":"Vaccines and immunotherapies that target peptide-major histocompatibility complexes (peptide-MHCs) have the potential to address multiple unmet medical needs in cancer and infectious disease. Designing vaccines and immunotherapies to target peptide-MHCs requires accurate identification of target peptides in infected or cancerous cells or tissue, and may require absolute or relative quantification to identify abundant targets and measure changes in presentation under different treatment conditions. Internal standard parallel reaction monitoring (also known as 'SureQuant') can be used to validate and/or quantify MHC peptides previously identified by using untargeted methods such as data-dependent acquisition. SureQuant MHC has three main use cases: (i) conclusive confirmation of the identities of putative MHC peptides via comparison with an internal synthetic stable isotope labeled (SIL) peptide standard; (ii) accurate relative quantification by using pre-formed heavy isotope-labeled peptide-MHC complexes (hipMHCs) containing SIL peptides as internal controls for technical variation; and (iii) absolute quantification of each target peptide by using different amounts of hipMHCs loaded with synthetic peptides containing one, two or three SIL amino acids to provide an internal standard curve. Absolute quantification can help determine whether the abundance of a peptide-MHC is sufficient for certain therapeutic modalities. SureQuant MHC therefore provides unique advantages for immunologists seeking to confidently validate antigenic targets and understand the dynamics of the MHC repertoire. After synthetic standards are ordered (3-4 weeks), this protocol can be carried out in 3-4 days and is suitable for individuals with mass spectrometry experience who are comfortable with customizing instrument methods.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504449","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

Visualizing plant salt stress with a NaCl-responsive fluorescent probe. 利用 NaCl 响应荧光探针观察植物盐胁迫。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-10-22 DOI: 10.1038/s41596-024-01068-x

Xiaoxie Ma, Xiaoyan Zeng, Yurou Huang, Sheng Hua Liu, Jun Yin, Guang-Fu Yang

{"title":"Visualizing plant salt stress with a NaCl-responsive fluorescent probe.","authors":"Xiaoxie Ma, Xiaoyan Zeng, Yurou Huang, Sheng Hua Liu, Jun Yin, Guang-Fu Yang","doi":"10.1038/s41596-024-01068-x","DOIUrl":"https://doi.org/10.1038/s41596-024-01068-x","url":null,"abstract":"Salt stress is an adverse environmental condition that harms plant growth and development. The development of salt stress probes is critical for tracking the growth dynamics of plants, molecular breeding or screening of growth regulators. The sodium chloride (NaCl)-responsive fluorescent probe Aza-CyBz is designed based on the tenet that NaCl induces formation of ordered aggregates, and the sensitive fluorescence response can enable the visualization of plant salt stress in root tip tissues and live plants. Herein, we describe a detailed three-step route for synthesis of Aza-CyBz and applications to monitoring salt stress in Arabidopsis thaliana. The procedures for operating fluorescence imaging under various stresses are also listed to eliminate interference from the oxidative mechanism of salt stress. Compared with conventional invasive approaches such as inductively coupled plasma emission spectrometry and flame photometer, our protocol can real-time monitor salt stress experienced by plants, which demands simple pretreatment procedure and staining technique. Due to near infrared fluorescence, this method provides direct visual observation of salt stress at both tissue and live plant levels, which is superior to conventional noninvasive approaches. The preparation of probe Aza-CyBz takes ~2 d, and the imaging experiments for assessing salt stress experienced by plants, including the preparation of stressed plant samples takes ~9-11 d for root tip tissues and ~23 d for live plants. Notably, acquisition and analysis visual images of salt stress in plants can be completed within 2 h and they require only a basic knowledge of spectroscopy and chemistry.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142504450","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 mouse model of volumetric muscle loss and therapeutic scaffold implantation. 肌肉体积损失和治疗性支架植入的小鼠模型。

IF 4.1 1区生物学

Nature Protocols Pub Date : 2024-10-18 DOI: 10.1038/s41596-024-01059-y

Caroline Hu, Gladys Chiang, Alex H-P Chan, Cynthia Alcazar, Karina H Nakayama, Marco Quarta, Thomas A Rando, Ngan F Huang

引用次数: 0

Measuring carbohydrate recognition profile of lectins on live cells using liquid glycan array (LiGA). 利用液态聚糖阵列（LiGA）测量活细胞上凝集素的碳水化合物识别特征。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-10-16 DOI: 10.1038/s41596-024-01070-3

Mirat Sojitra, Edward N Schmidt, Guilherme M Lima, Eric J Carpenter, Kelli A McCord, Alexey Atrazhev, Matthew S Macauley, Ratmir Derda

{"title":"Measuring carbohydrate recognition profile of lectins on live cells using liquid glycan array (LiGA).","authors":"Mirat Sojitra, Edward N Schmidt, Guilherme M Lima, Eric J Carpenter, Kelli A McCord, Alexey Atrazhev, Matthew S Macauley, Ratmir Derda","doi":"10.1038/s41596-024-01070-3","DOIUrl":"https://doi.org/10.1038/s41596-024-01070-3","url":null,"abstract":"Glycans constitute a significant fraction of biomolecular diversity on cellular surfaces across all kingdoms of life. As the structure of glycans is not directly encoded by the organism's DNA, it is impossible to use high-throughput DNA technologies to study the role of cellular glycosylation or to understand how glycocalyx is recognized by glycan-binding proteins (GBPs). To address this gap, we recently described a liquid glycan array (LiGA) platform that allows profiling of glycan-GBP interactions on the surface of live cells in vitro and in vivo using next-generation sequencing. LiGA is a library of DNA-barcoded bacteriophages, where each clonal bacteriophage displays 5-1,500 copies of a glycan and the distinct DNA barcode inside each bacteriophage clone encodes the structure and density of the displayed glycans. Deep sequencing of the glycophages associated with live cells yields a glycan-binding profile of GBPs expressed on the surface of cells. This protocol provides detailed instructions for how to use LiGA to probe cell surface receptors and includes information on the preparation of glycophages, analysis by MALDI-TOF mass spectrometry, the assembly of a LiGA library and its deep sequencing. Using this protocol, we measure glycan-binding profiles of the immunomodulatory sialic acid-binding immunoglobulin-like lectins‑1, -2, -6, -7 and -9 expressed on the surface of different cell types. Compared with existing methods that require complex specialist equipment, this method allows users with basic molecular biology expertise to measure the precise glycan-binding profile of GBPs on the surface of any cell type expressing exogenous GBP within 2-3 d.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470349","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

Publisher Correction: Generation of 'semi-guided' cortical organoids with complex neural oscillations. 出版商更正：生成具有复杂神经振荡的 "半引导 "皮层有机体

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-10-15 DOI: 10.1038/s41596-024-01087-8

Michael Q Fitzgerald, Tiffany Chu, Francesca Puppo, Rebeca Blanch, Miguel Chillón, Shankar Subramaniam, Alysson R Muotri

引用次数: 0

Easy and accurate protein structure prediction using ColabFold. 使用 ColabFold 轻松准确地预测蛋白质结构。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-10-14 DOI: 10.1038/s41596-024-01060-5

Gyuri Kim, Sewon Lee, Eli Levy Karin, Hyunbin Kim, Yoshitaka Moriwaki, Sergey Ovchinnikov, Martin Steinegger, Milot Mirdita

{"title":"Easy and accurate protein structure prediction using ColabFold.","authors":"Gyuri Kim, Sewon Lee, Eli Levy Karin, Hyunbin Kim, Yoshitaka Moriwaki, Sergey Ovchinnikov, Martin Steinegger, Milot Mirdita","doi":"10.1038/s41596-024-01060-5","DOIUrl":"https://doi.org/10.1038/s41596-024-01060-5","url":null,"abstract":"Since its public release in 2021, AlphaFold2 (AF2) has made investigating biological questions, by using predicted protein structures of single monomers or full complexes, a common practice. ColabFold-AF2 is an open-source Jupyter Notebook inside Google Colaboratory and a command-line tool that makes it easy to use AF2 while exposing its advanced options. ColabFold-AF2 shortens turnaround times of experiments because of its optimized usage of AF2's models. In this protocol, we guide the reader through ColabFold best practices by using three scenarios: (i) monomer prediction, (ii) complex prediction and (iii) conformation sampling. The first two scenarios cover classic static structure prediction and are demonstrated on the human glycosylphosphatidylinositol transamidase protein. The third scenario demonstrates an alternative use case of the AF2 models by predicting two conformations of the human alanine serine transporter 2. Users can run the protocol without computational expertise via Google Colaboratory or in a command-line environment for advanced users. Using Google Colaboratory, it takes <2 h to run each procedure. The data and code for this protocol are available at https://protocol.colabfold.com .","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470348","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

kallisto, bustools and kb-python for quantifying bulk, single-cell and single-nucleus RNA-seq. kallisto、bustools 和 kb-python，用于量化批量、单细胞和单核 RNA-seq。

IF 13.1 1区生物学

Nature Protocols Pub Date : 2024-10-10 DOI: 10.1038/s41596-024-01057-0

Delaney K Sullivan, Kyung Hoi Joseph Min, Kristján Eldjárn Hjörleifsson, Laura Luebbert, Guillaume Holley, Lambda Moses, Johan Gustafsson, Nicolas L Bray, Harold Pimentel, A Sina Booeshaghi, Páll Melsted, Lior Pachter

{"title":"kallisto, bustools and kb-python for quantifying bulk, single-cell and single-nucleus RNA-seq.","authors":"Delaney K Sullivan, Kyung Hoi Joseph Min, Kristján Eldjárn Hjörleifsson, Laura Luebbert, Guillaume Holley, Lambda Moses, Johan Gustafsson, Nicolas L Bray, Harold Pimentel, A Sina Booeshaghi, Páll Melsted, Lior Pachter","doi":"10.1038/s41596-024-01057-0","DOIUrl":"10.1038/s41596-024-01057-0","url":null,"abstract":"The term 'RNA-seq' refers to a collection of assays based on sequencing experiments that involve quantifying RNA species from bulk tissue, single cells or single nuclei. The kallisto, bustools and kb-python programs are free, open-source software tools for performing this analysis that together can produce gene expression quantification from raw sequencing reads. The quantifications can be individualized for multiple cells, multiple samples or both. Additionally, these tools allow gene expression values to be classified as originating from nascent RNA species or mature RNA species, making this workflow amenable to both cell-based and nucleus-based assays. This protocol describes in detail how to use kallisto and bustools in conjunction with a wrapper, kb-python, to preprocess RNA-seq data. Execution of this protocol requires basic familiarity with a command line environment. With this protocol, quantification of a moderately sized RNA-seq dataset can be completed within minutes.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142400787","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