Nature Protocols最新文献

{"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":"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. This protocol uses the liquid glycan array (LiGA) platform, a library of DNA-barcoded bacteriophages displaying 5–1,500 copies of a glycan, to allow profiling of glycan–glycan-binding protein interactions on the surface of live cells.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 4","pages":"989-1019"},"PeriodicalIF":13.1,"publicationDate":"2024-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.nature.com/articles/s41596-024-01070-3.pdf","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":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Publisher Correction: Generation of 'semi-guided' cortical organoids with complex neural oscillations.","authors":"Michael Q Fitzgerald, Tiffany Chu, Francesca Puppo, Rebeca Blanch, Miguel Chillón, Shankar Subramaniam, Alysson R Muotri","doi":"10.1038/s41596-024-01087-8","DOIUrl":"https://doi.org/10.1038/s41596-024-01087-8","url":null,"abstract":"","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":" ","pages":""},"PeriodicalIF":13.1,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142470350","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":"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":"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 . We describe the use of ColabFold to perform structure prediction of monomers, complexes and alternative conformations, either on the web or locally, and provide guidance on interpreting the results through confidence metrics and visualizations.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 3","pages":"620-642"},"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}

{"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. Kallisto, bustools and kb-python are a set of tools for quantifying bulk, single-cell and single-nucleus RNA-seq. Together, this set of free, open-source software tools can produce gene expression quantification from raw sequencing reads.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 3","pages":"587-607"},"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}

{"title":"Open-vessel polymerization of N-carboxyanhydride (NCA) for polypeptide synthesis","authors":"Yueming Wu, Kang Chen, Jiangzhou Wang, Wenhui Dai, Haowen Yu, Xinyi Xie, Minzhang Chen, Runhui Liu","doi":"10.1038/s41596-024-01062-3","DOIUrl":"10.1038/s41596-024-01062-3","url":null,"abstract":"Synthetic polypeptides, also known as poly(α-amino acids), have the same polyamide backbone structures as natural proteins and peptides. As an important class of biomaterials, polypeptides have been widely used because of their biocompatibility, bioactivity and biodegradability. Ring-opening polymerization of N-carboxyanhydride (NCA) is a classical and widely used method for the synthesis of polypeptides. The dominantly used primary amine–initiated NCA polymerization can yield well-defined polymers and complex macromolecular architectures, but the reaction is slow and sensitive to moisture, making it necessary to use anhydrous solvents and a glovebox. One solution is to use lithium hexamethyldisilazide (LiHMDS) as the initiator, as described in this protocol. LiHMDS-initiated NCA polymerization is less sensitive to moisture and can be carried out in an open vessel outside the glovebox. It is also very fast; the reaction can be complete within 5 min to produce 30-mer polypeptides. In this protocol, poly(γ-benzyl-l-glutamate) is prepared as an example, but the protocol can easily be adapted to the synthesis of other polypeptides by generating NCAs from different amino acids, making it particularly suitable for the efficient parallel synthesis of polypeptide libraries. We provide detailed procedures for NCA synthesis and purification, the method of polymer end-group modification and measurement of polymerization kinetics and reactivity ratio. The procedure for synthesis of monomers and polymerization to form polypeptides requires <1 d. The superfast and open-vessel NCA polymerization method described here will probably enable a wide range of applications in the synthesis and functional study of polypeptide biomaterials. Synthetic polypeptides are used in many applications in which biocompatibility is important. This protocol describes a fast lithium hexamethyldisilazide-initiated N-carboxyanhydride ring-opening polymerization method for their synthesis.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 3","pages":"709-726"},"PeriodicalIF":13.1,"publicationDate":"2024-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391835","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":"How to isolate channel-forming membrane proteins using the E. coli expression system","authors":"Claudio Piselli","doi":"10.1038/s41596-024-01055-2","DOIUrl":"10.1038/s41596-024-01055-2","url":null,"abstract":"The recombinant expression, isolation and characterization of pore-forming proteins is one of the most commonly used strategies for understanding the permeability properties of the biological membrane into which they are embedded. This protocol describes how to quantify the expression of your protein of interest and use this information to optimize its production using the Escherichia coli strain BL21Gold(de3)ΔABCF. It explains with a step-by-step approach how to separate the bacterial compartments according to their solubility and how to extract your protein of interest in its native conformation using detergent solutions. Finally, it describes how to improve its purity via ion-exchange chromatography and insert the purified porins into outer membrane vesicles, from which they can be copurified. The protocol is simpler and less empirical than those described for most channel-forming membrane proteins and also provides a solid foundation for the isolation of soluble proteins. Several parameters can be optimized on a case-by-case basis: expression time and temperature, concentration of the inducer, nature and concentration of the detergent, incubation time and temperature, pH and ionic strength of the purification buffers. This protocol is effective with prokaryotic channel-forming membrane proteins and can be employed for the production of pore-forming proteins from chloroplasts, mitochondria or eukaryotes in general. With minor optimization, this protocol can be adapted for the isolation of receptors, carrier, pumps or any other membrane-active proteins. Channel-forming membrane proteins control solute exchange across membranes, and they are difficult to obtain with sufficient yield and purity. This protocol describes a universal approach to their expression (in Escherichia coli), extraction and purification.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 2","pages":"462-479"},"PeriodicalIF":13.1,"publicationDate":"2024-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142375652","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":"High-confidence and high-throughput quantification of synapse engulfment by oligodendrocyte precursor cells","authors":"Jessica A. Kahng, Andre M. Xavier, Austin Ferro, Samantha X. Tang, Yohan S. S. Auguste, Lucas Cheadle","doi":"10.1038/s41596-024-01048-1","DOIUrl":"10.1038/s41596-024-01048-1","url":null,"abstract":"Oligodendrocyte precursor cells (OPCs) sculpt neural circuits through the phagocytic engulfment of synapses during development and adulthood. However, existing techniques for analyzing synapse engulfment by OPCs have limited accuracy. Here we describe the quantification of synapse engulfment by OPCs via a two-pronged cell biological approach that combines high-confidence and high-throughput methodologies. Firstly, an adeno-associated virus encoding a pH-sensitive, fluorescently tagged synaptic marker is expressed in neurons in vivo to differentially label presynaptic inputs, depending upon whether they are outside of or within acidic phagolysosomal compartments. When paired with immunostaining for OPC markers in lightly fixed tissue, this approach quantifies the engulfment of synapses by around 30–50 OPCs in each experiment. The second method uses OPCs isolated from dissociated brain tissue that are then fixed, incubated with fluorescent antibodies against presynaptic proteins, and analyzed by flow cytometry, enabling the quantification of presynaptic material within tens of thousands of OPCs in <1 week. The integration of both methods extends the current imaging-based assays, originally designed to quantify synaptic phagocytosis by other brain cells such as microglia and astrocytes, by enabling the quantification of synaptic engulfment by OPCs at individual and populational levels. With minor modifications, these approaches can be adapted to study synaptic phagocytosis by numerous glial cell types in the brain. The protocol is suitable for users with expertise in both confocal microscopy and flow cytometry. The imaging-based and flow cytometry-based protocols require 5 weeks and 2 d to complete, respectively. An approach that combines a pH-sensitive synaptic biomarker expressed in vivo with the ex vivo staining of oligodendrocyte precursor cells enables quantification of synapse engulfment by oligodendrocyte precursor cells at single-cell and population-level resolution.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 2","pages":"407-439"},"PeriodicalIF":13.1,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142372351","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":"Multiplexed chromatin immunoprecipitation sequencing for quantitative study of histone modifications and chromatin factors","authors":"Banushree Kumar, Carmen Navarro, Philip Yuk Kwong Yung, Jing Lyu, Angelo Salazar Mantero, Anna-Maria Katsori, Hannah Schwämmle, Marcel Martin, Simon J. Elsässer","doi":"10.1038/s41596-024-01058-z","DOIUrl":"10.1038/s41596-024-01058-z","url":null,"abstract":"ChIP–seq is a widely used technique for studying histone post-translational modifications and DNA-binding proteins. DNA fragments associated with a specific protein or histone modification epitope are captured by using antibodies, sequenced and mapped to a reference genome. Albeit versatile and popular, performing many parallel ChIP–seq experiments to compare different conditions, replicates and epitopes is laborious, is prone to experimental variation and does not allow quantitative comparisons unless adequate spike-in chromatin is included. We present a detailed protocol for performing and analyzing a multiplexed quantitative chromatin immunoprecipitation-sequencing experiment (MINUTE-ChIP), in which multiple samples are profiled against multiple epitopes in a single workflow. Multiplexing not only dramatically increases the throughput of ChIP–seq experiments (e.g., profiling 12 samples against multiple histone modifications or DNA-binding proteins in a single experiment), but also enables accurate quantitative comparisons. The protocol consists of four parts: sample preparation (i.e., lysis, chromatin fragmentation and barcoding of native or formaldehyde-fixed material), pooling and splitting of the barcoded chromatin into parallel immunoprecipitation reactions, preparation of next-generation sequencing libraries from input and immunoprecipitated DNA and data analysis using our dedicated analysis pipeline. This pipeline autonomously generates quantitatively scaled ChIP–seq tracks for downstream analysis and visualization, alongside necessary quality control indicators. The entire workflow requires basic knowledge in molecular biology and bioinformatics and can be completed in 1 week. MINUTE-ChIP empowers biologists to perform every ChIP–seq experiment with an appropriate number of replicates and control conditions, delivering more statistically robust, exquisitely quantitative and biologically meaningful results. MINUTE-ChIP is a multiplexed chromatin immunoprecipitation and sequencing method that measures global and locus-specific changes in histone modification patterns and chromatin factor binding across multiple samples and conditions.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 3","pages":"779-809"},"PeriodicalIF":13.1,"publicationDate":"2024-10-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142372352","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":"Plant sperm cell sequencing for genome phasing and determination of meiotic crossover points","authors":"Weiyi Zhang, Arslan Tariq, Xinxin Jia, Jianbing Yan, Alisdair R. Fernie, Björn Usadel, Weiwei Wen","doi":"10.1038/s41596-024-01063-2","DOIUrl":"10.1038/s41596-024-01063-2","url":null,"abstract":"Haplotype phasing represents a pivotal procedure in genome analysis, entailing the identification of specific genetic variant combinations on each chromosome. Achieving chromosome-level genome phasing constitutes a considerable challenge, particularly in organisms with large and complex genomes. To address this challenge, we have developed a robust, gamete cell-based phasing pipeline, including wet-laboratory processes for plant sperm cell isolation, short-read sequencing and a bioinformatics workflow to generate chromosome-level phasing. The bioinformatics workflow is applicable for both plant and other sperm cells, for example, those of mammals. Our pipeline ensures high-quality single-nucleotide polymorphism (SNP) calling for each sperm cell and the subsequent construction of a high-density genetic map. The genetic map facilitates accurate chromosome-level genome phasing, enables crossover event detection and could be used to correct potential assembly errors. Our bioinformatics pipeline runs on a Linux system and most of its steps can be executed in parallel, expediting the analysis process. The entire workflow can be performed over the course of 1 d. We provide a practical example from our previous research using this protocol and provide the whole bioinformatics pipeline as a Docker image to ensure its easy adaptability to other studies. This protocol describes a method for haplotype phasing plant genomes, using gamete cells to enable chromosome-level phasing and crossover detection without the need for Hi-C data or sequencing of large plant populations.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 3","pages":"690-708"},"PeriodicalIF":13.1,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365856","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":"Use of synthetic circular RNA spike-ins (SynCRS) for normalization of circular RNA sequencing data","authors":"Vanessa M. Conn, Ryan Liu, Marta Gabryelska, Simon J. Conn","doi":"10.1038/s41596-024-01053-4","DOIUrl":"10.1038/s41596-024-01053-4","url":null,"abstract":"High-throughput RNA sequencing enables the quantification of transcript abundance and the identification of novel transcripts in biological samples. These include circular RNAs (circRNAs), a family of alternatively spliced RNA molecules that form a continuous loop. However, quantification and comparison of circRNAs between RNA sequencing libraries remain challenging due to confounding errors introduced during exonuclease digestion, library preparation and RNA sequencing itself. Here we describe a set of synthetic circRNA spike-ins—termed ‘SynCRS’—that can be added directly to purified RNA samples before exonuclease digestion and library preparation. SynCRS, introduced either individually or in combinations of varying size and abundance, can be integrated into all next-generation sequencing workflows and, critically, facilitate the quantitative calibration of circRNA transcript abundance between samples, tissue types, species and laboratories. Our step-by-step protocol details the generation of SynCRS and guides users on the stoichiometry of SynCRS spike-in to RNA samples, followed by the bioinformatic steps required to facilitate quantitative comparisons of circRNAs between libraries. The laboratory steps to produce the SynCRS require an additional 3 d on top of the high throughput circRNA sequencing and bioinformatics. The protocol is suitable for users with basic experience in molecular biology and bioinformatics. This protocol presents a method for calibrating circular RNA abundance for comparison between RNA sequencing libraries, utilizing a spike-in of synthetic circular RNAs.","PeriodicalId":18901,"journal":{"name":"Nature Protocols","volume":"20 2","pages":"387-406"},"PeriodicalIF":13.1,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142350444","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}