Current protocols最新文献

{"title":"Morphogen-Directed, High-Throughput Development of hiPSC-Derived Telencephalic Organoids for Comparative In Vitro Phenotyping","authors":"Dosh Whye,&nbsp;Erika M. Norabuena,&nbsp;Wardiya Afshar-Saber,&nbsp;Becca Lewis,&nbsp;Taryn J. Polanco,&nbsp;Delaney Wood,&nbsp;Mustafa Sahin,&nbsp;Elizabeth D. Buttermore","doi":"10.1002/cpz1.70449","DOIUrl":"10.1002/cpz1.70449","url":null,"abstract":"<p>Pluripotent stem cell (PSC)–derived telencephalic organoids have long been established as an <i>in vitro</i> cell model to study the molecular and cellular features of the developing cerebral cortex and associated brain structures. Numerous directed organoid differentiation protocols have been developed that recapitulate cell subtype specification, cytoarchitectural organization, and cellular function with a high degree of fidelity. Protocol development has focused on the scalability of this <i>in vitro</i> system, and most organoid culture platforms involve a population-based approach in large vessel sizes. This makes comparative study with multiple cell lines both costly and time-consuming, and lowers the throughput for phenotypic screening. We have established a high-throughput approach for <i>in vitro</i> generation of hiPSC-derived telencephalic organoids that can be used to develop comparative phenotypic assays involving high-content imaging platforms. This article first highlights the use of a scaled-down, miniaturized cell culture volume that is amenable to high-throughput production of multiple 384-well plates. Then, using controlled morphogen cues, 3D hiPSC aggregates are directed into an anterior telencephalic identity and further specified into dorsal pallium or ventral subpallium cell fates, which can additionally be combined into assembloids for modeling excitatory and inhibitory networks or left to develop separately as single organoid subtypes. Finally, we introduce a Support Protocol detailing an <i>in vitro</i> 3D cell painting assay for organoid morpho-phenotypic characterization. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: High-throughput 3D aggregation of hiPSCs in morphogen-rich media</p><p><b>Basic Protocol 2</b>: Recombinant inhibitory protein–directed induction of telencephalic organoids</p><p><b>Basic Protocol 3</b>: Morphogen-guided induction of dorsal telencephalic/pallial organoids</p><p><b>Basic Protocol 4</b>: Morphogen-guided induction of ventral telencephalic/subpallial organoids</p><p><b>Support Protocol</b>: 3D live cell painting for organoid morphometric phenotyping</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148984157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Embryotoxicity and Cardiotoxicity Assessment of Chemical Compounds Using Eggs of Lymnaea stagnalis","authors":"Kesavi Sri Malika Maddila,&nbsp;Meghana Korikani,&nbsp;Rumana Ansari,&nbsp;Rida Fatima,&nbsp;Preetha Kemphu,&nbsp;Rama Krishna Kancha","doi":"10.1002/cpz1.70457","DOIUrl":"https://doi.org/10.1002/cpz1.70457","url":null,"abstract":"<p>There is a paucity of invertebrate models for the assessment of general and organ-specific toxicity caused by chemicals, such as chemotherapeutics and environmental pollutants. To address this, we have established the embryo of <i>Lymnaea stagnalis</i> (great pond snail) as a simple and rapid model to assay toxicity in a medium-throughput manner. For measuring general toxicity, we have successfully repurposed Alamar Blue dye to assay physiological activity of the embryos. Due to the transparent nature of the <i>L. stagnalis</i> eggs, we have employed a microscope-based phenotypic assay to determine cardiotoxicity. Furthermore, we have previously shown that this model can be used to test cardioprotective strategies to overcome toxicity indued by cancer chemotherapeutics. Due to similarities of the <i>L. stagnalis</i> heart with that of a vertebrate heart, combined with the advantages of easy handling, <i>L. stagnalis</i> eggs serve as an emerging model for embryotoxicity and cardiotoxicity screening of pharmaceutical agents and environmental pollutants. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Isolation of clusters and separation of individual eggs of <i>L. stagnalis</i></p><p><b>Basic Protocol 2</b>: Assessment of cardiotoxicity and evaluation of cardioprotective strategies</p><p><b>Basic Protocol 3</b>: Alamar Blue dye–based embryotoxicity test</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148943206","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Light-Activated Molecular Purification (LAMP): A Photo-Responsive Affinity-Chromatography Protocol for Recombinant Protein Purification","authors":"Krishna Agrawal,&nbsp;Aishwarya Satyanarayana,&nbsp;Yashwant Kumar,&nbsp;Karthik Pushpavanam","doi":"10.1002/cpz1.70440","DOIUrl":"https://doi.org/10.1002/cpz1.70440","url":null,"abstract":"<p>Recombinant protein purification is a critical step prior to their use in therapeutics and diagnostics. Affinity and ion-exchange chromatography are among the most widely used purification strategies; however, both rely on extensive trial-and-error optimization and require chemical additives or high salt concentrations for protein elution. These conditions necessitate additional, time-consuming downstream dialysis steps to remove residual chemicals. To address these limitations, we propose a light-triggered protein elution strategy that eliminates the need for chemical elution agents and simplifies downstream processing. This approach exploits PhoCl1, a photocleavable protein that undergoes light-induced cleavage into an N-terminal empty barrel and a short C-terminal peptide fragment (Zhang et al., 2017; Lu et al., 2021). We engineered a fusion construct in which a 6×His tag is positioned at the N-terminus of PhoCl1, while the protein of interest (POI) is fused to its C-terminus (6×His–PhoCl1–POI). Following immobilization of the whole-cell lysate on Ni-NTA resin and removal of nonspecifically bound proteins, light exposure induces PhoCl1 cleavage. The His-tagged N-terminal fragment remains bound to the resin, while the POI is selectively released into the supernatant. Using this method, we have achieved protein purities exceeding 90%, demonstrating a rapid, reagent-free, and highly efficient alternative to conventional protein purification workflows. Here, for demonstration purposes, we have included a POI, riboflavin kinase (<i>Mj</i>RibK) found in <i>Methanocaldococcus jannaschii</i>. In this protocol, we describe the design, expression, and characterization of PhoCl1 fusion proteins using an intracellular expression system in <i>Escherichia coli</i>, with all steps optimized for cytosolic production. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Light-Activated Affinity-Based Recombinant Protein Purification</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148943023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Step-by-Step Guide to Successful Multiparametric Microbiota Flow Cytometry","authors":"Toni Sempert,&nbsp;Lisa Budzinski,&nbsp;Robin Kempkens,&nbsp;Gi-Ung Kang,&nbsp;René Maier,&nbsp;Hyun-Dong Chang","doi":"10.1002/cpz1.70460","DOIUrl":"10.1002/cpz1.70460","url":null,"abstract":"<p>The role of the human microbiota in host health is an important area of research. Conventional characterization of the complex microbial communities colonizing the human body is primarily performed using culture-based or high-throughput sequencing approaches, which have improved general understanding yet lack cellular features relevant for interaction and function at the single-cell level. Flow cytometry is an established tool for single-cell analysis, and recent improvements, such as increased resolution of small cells and particles, offer possibilities for characterizing complex microbial communities. Here, we describe an updated protocol to characterize complex microbial communities derived from human stool samples at the single-cell level by multiparametric microbiota flow cytometry. Our protocol covers the process from the isolation of bacteria from stool, preparation of cryo-stocks, staining procedure for phenotypic features of the bacteria, and flow cytometric analysis. Our phenotypic characterization includes light scattering properties, quantitative DNA staining, isotype-specific staining of host–antibody coating, and profiling of bacterial cell surface sugar moieties. In addition, we highlight the importance of determining the bacterial load for improved biological interpretation. External staining controls ensure reproducibility and quality controls. Additionally, we suggest a downstream analysis pipeline involving segmentation of multivariate data by a self-organized map (SOM) and machine learning to generate specific microbiota fingerprints. Optionally, bacteria with specific phenotypic characteristics can be isolated by fluorescence-activated cell sorting (FACS) for further investigation. Our protocol can be applied to any microbial community or sample source and offers the flexibility to be expanded with additional phenotypic markers. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of cryo-preserved bacterial stocks from a single-cell microbiota suspension derived from native human stool sample</p><p><b>Basic Protocol 2</b>: Staining protocol for cryo-preserved microbiota stocks and acquisition using a flow cytometer</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13544355/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148893170","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimized Extraction Protocol of Chloroplast DNA from Chenopodium album (Amaranthaceae) Enabling Whole Plastid Genome Sequencing.","authors":"Rajkumari Jashmi Devi, Merinashwari Konjengbam, Biseshwori Thongam","doi":"10.1002/cpz1.70482","DOIUrl":"https://doi.org/10.1002/cpz1.70482","url":null,"abstract":"<p><p>Chenopodium is one of the most complex genera in the family Amaranthaceae, exhibiting ploidy levels ranging from diploid to hexaploid. The lack of distinct morphological characters and phenotypic plasticity has complicated species identification in this genus. The advent of next-generation sequencing technology has enabled in-depth genomic studies of Chenopodium species. To date, the chloroplast genomes of six Chenopodium species-Chenopodium acuminatum, C. ficifolium, C. berlandieri, C. petiolare, C. quinoa, and C. album-have been reported using genomic DNA as templates. On the other hand, high-quality chloroplast DNA (cpDNA) is recommended for effective chloroplast genome sequencing. The present study reports an optimized high-salt extraction method for extracting cpDNA from C. album. The quality of isolated cpDNA was validated by determining the complete chloroplast genome of C. album using the Illumina platform with a 150 × 2-pair-end (PE) library and cpDNA as the template. The chloroplast genome of C. album is 152,028 base pairs and contains 129 genes. Of these, 84 are protein-coding genes, 8 are rRNA genes, and 37 are tRNA genes. This study lays the foundation for isolating high-quality cpDNA using a simple yet efficient method for extracting cpDNA from C. album, enabling efficient sequencing of the whole plastid genome for further understanding of the evolution, genetics, and taxonomy of this complex plant. © 2026 Wiley Periodicals LLC Basic Protocol: An optimized high-salt extraction protocol for chloroplast DNA isolation from Chenopodium album (Amaranthaceae), enabling whole plastid genome sequencing.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":"e70482"},"PeriodicalIF":2.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149066907","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A Stepwise Method to Derive Functional Pancreatic Islet-like Clusters from Human Pluripotent Stem Cells.","authors":"Manuj Bandral, Jimena Alvarez-Castanon, David S Lorberbaum","doi":"10.1002/cpz1.70467","DOIUrl":"10.1002/cpz1.70467","url":null,"abstract":"<p><p>The most common treatment for people with people with type 1 diabetes (T1D) requires injections of exogenous insulin to regulate blood glucose homeostasis. A longer-term therapeutic solution for T1D has emerged whereby patients may regain insulin independence through transplantation of donated human pancreatic islets, which contain insulin-secreting beta (β)-cells. Donated islets, however, are in short supply. Much effort has been dedicated to using human pluripotent stem cell (hPSC)-derived islets (SC-islets) as an unlimited source of insulin-secreting β-cells. Here, we describe a robust and reproducible 40-day, seven-stage stepwise process for generating hPSC-derived SC-islets. Our differentiation protocol aims to mimic in vivo pancreas development using stage-specific signals to promote the generation of SC-islets. The initial maintenance and the first four stages are performed in two-dimensional monolayer culture; the subsequent three stages require three-dimensional culture in microwells to induce the formation of islet-like clusters. The SC-islets generated at the end of the procedure respond to glucose challenges, reliably express appropriate endocrine markers, contain intracellular insulin, and display an islet-like architecture. Most SC-islets also partially express maturity markers that are often challenging to activate in vitro. These protocols thus provide a scalable platform for generating SC-islets for disease modeling and cell replacement studies. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Thawing, recovery, and passaging of cryopreserved hPSCs Support Protocol 1: Preparation of solutions and media for hPSC maintenance and expansion Support Protocol 2: Preparation of Matrigel-coated plates for passaging hSPCs Basic Protocol 2: Differentiation of hPSCs to SC-islets.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":"e70467"},"PeriodicalIF":2.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13614477/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149066839","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Using the Job Dispatcher Web Interfaces for Sequence Analysis.","authors":"Nandana Madhusoodanan, Fábio Madeira, Ania Niewielska, Tariq Sulaiman Fazil Ahamed, Leeroy M Ncube, Timothy Porter","doi":"10.1002/cpz1.70416","DOIUrl":"10.1002/cpz1.70416","url":null,"abstract":"<p><p>The rapid growth of biological sequence data has increased the need for easy, accessible, and scalable tools for sequence comparison and analysis. The EMBL-EBI Job Dispatcher framework helps meet this need by providing a single web-based platform that offers many commonly used tools, including the BLAST, Clustal Omega, and EMBOSS programs. In this article, we provide a practical, easy-to-follow guide to the main tool categories available in Job Dispatcher, focusing on multiple sequence alignment and sequence similarity search. We present step-by-step workflows that demonstrate how to run these analyses directly on the web without installing any bioinformatics software. Our goal is to provide a simple, practical reference for researchers and students new to bioinformatics, helping them choose the right tools and use web-based analysis effectively. © 2026 EMBL-EBI. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sequence similarity search using FASTA Basic Protocol 2: Sequence similarity search using NCBI BLAST+ Basic Protocol 3: Sequence similarity search using PSI-BLAST Basic Protocol 4: Multiple sequence alignment using Clustal Omega Basic Protocol 5: Visualizing multiple sequence alignments using MView Basic Protocol 6: Workflow from BLAST results to Clustal Omega to MView.</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":"e70416"},"PeriodicalIF":2.5,"publicationDate":"2026-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13615794/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149066921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Sequence-Specific Primer Polymerase Chain Reaction Genotyping of the Human IL33 Polymorphism rs1929992 (T > C) with Sanger Sequencing Validation","authors":"Aléia Harumi Uchibaba Yamanaka,&nbsp;Guilherme Castellani,&nbsp;Matheus Braga,&nbsp;Josiane Bazzo de Alencar,&nbsp;Letícia Cristina de Almeida Silva,&nbsp;Quirino Alves de Lima Neto","doi":"10.1002/cpz1.70455","DOIUrl":"10.1002/cpz1.70455","url":null,"abstract":"<p>Single-nucleotide polymorphisms (SNPs) may influence gene expression and contribute to interindividual variability in immune responses and disease susceptibility. Interleukin-33 (IL33), a cytokine involved in inflammation, immune regulation, epithelial barrier homeostasis, and tissue remodeling, has been implicated in allergic, autoimmune, inflammatory, and neoplastic disorders. The <i>IL33</i> rs1929992 (T &gt; C) polymorphism, located in intron 3, has been associated with altered IL33 expression, with the T allele linked to increased cytokine production compared with the C allele. This article describes a standardized sequence-specific primer polymerase chain reaction (PCR-SSP) protocol for genotyping the human <i>IL33</i> rs1929992 polymorphism, with validation by Sanger sequencing. The protocol includes optimization of annealing temperature, MgCl₂ concentration, primer balance, deoxynucleotide triphosphates (dNTPs), and DNA input to ensure amplification specificity and reproducibility. Allele-specific amplification is achieved using forward primers specific for the T and C alleles in independent reactions and analyzed by agarose gel electrophoresis, with the <i>human growth hormone</i> (<i>HGH</i>) gene used as an internal control. This protocol provides a simple, reproducible, and accessible method for rs1929992 genotyping, suitable for population-based and genetic association studies involving immune-mediated and inflammatory diseases. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: PCR-SSP genotyping of <i>IL33</i> rs1929992</p><p><b>Support Protocol 1</b>: Extraction of genomic DNA by salting-out</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13528966/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148868296","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Massively Parallel Profiling of Single-Cell RNA Dynamics Using Well-TEMP-seq","authors":"Di Wang,&nbsp;Qiqi Lv,&nbsp;Shichao Lin","doi":"10.1002/cpz1.70458","DOIUrl":"10.1002/cpz1.70458","url":null,"abstract":"<p>Single-cell RNA sequencing (scRNA-seq) reveals the transcriptional heterogeneity of cells, revolutionizing our understanding of cellular processes. However, the static snapshots obtained from scRNA-seq fail to reveal the time-resolved dynamics of transcription, which impedes critical insights into various biological processes, such as cellular differentiation, embryonic development, disease progression, and responses to external stimuli. Here, we describe Well-TEMP-seq, a protocol for massively parallel profiling of the temporal dynamics of single-cell gene expression. Well-TEMP-seq combines metabolic RNA labeling with a microwell-based scRNA-seq method, Well-paired-seq, to distinguish newly transcribed RNAs marked by T-to-C substitutions from pre-existing RNAs in each of thousands of single cells. Well-TEMP-seq is high-throughput, cost-effective, accurate, and provides a low cell loss rate and high single cell/bead pairing efficiency. More importantly, Well-TEMP-seq can be easily set up in other labs, and the loading of cells and beads can be easily accomplished by an optical microscope and a pipette. We believe that Well-TEMP-seq will be widely adopted and help researchers perform transformative research to unveil the dynamics of single-cell gene expression in diverse biological processes. © 2026 Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Well-paired-seq chip fabrication</p><p><b>Basic Protocol 2</b>: Well-TEMP-seq sample processing</p><p><b>Basic Protocol 3</b>: Bioinformatics analysis</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148868280","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacterial Lysis Observation Over Time Assay (BLOTS) for High-Throughput Isolation, Sequencing, and Characterization of Bacteriophages","authors":"Sean Jia Le Pang,&nbsp;Soon Keong Wee,&nbsp;Bobby Guo Wei Lee,&nbsp;Odelia Yap,&nbsp;Eric Peng Huat Yap","doi":"10.1002/cpz1.70454","DOIUrl":"10.1002/cpz1.70454","url":null,"abstract":"<p>Phages remain severely under-sampled, largely due to the limitations of current isolation methods, which are labor-intensive, low-throughput, and often restricted to a narrow set of bacterial hosts. To address this gap, we developed a robust and scalable field-to-genome workflow that leverages laboratory automation to screen environmental samples against a comprehensive 188-strain bacterial host library using a 384-well plate format. We demonstrate the workflow using <i>Acinetobacter baumannii</i> as the primary model host. Phage activity is detected through Bacterial Lysis Observation over Time assay (BLOTS) using spectrophotometry. Subsequently, isolation and subculture of the phages are done with a single layer agar spot assay. Phage genomes are then sequenced using long-read technology and annotated. This enables systematic and reproducible phage detection and enrichment from diverse sample types. The use of large numbers of host strains streamlines phage discovery and substantially increases the chances of isolating novel phages. The BLOTS high-throughput approach can also be used for screening phage collections against specified clinical hosts of interest for phage research and candidate selection for phage therapy applications. © 2026 The Author(s). <i>Current Protocols</i> published by Wiley Periodicals LLC.</p><p><b>Basic Protocol 1</b>: Preparation of host libraries and multi-host primary enrichment</p><p><b>Basic Protocol 2</b>: Preparation of the BLOTS screening plate using an automated liquid handler</p><p><b>Alternate Protocol 1</b>: Manual phage screening without laboratory automation</p><p><b>Basic Protocol 3</b>: Phage screening using BLOTS</p><p><b>Basic Protocol 4</b>: Phage validation using the single-layer agar spot assay</p><p><b>Basic Protocol 5</b>: Phage amplification for downstream applications</p><p><b>Basic Protocol 6</b>: Phage concentration using PEG-8000 precipitation</p><p><b>Alternate Protocol 2</b>: Phage concentration using centrifugal filtration</p><p><b>Basic Protocol 7</b>: Automated DNA extraction with magnetic beads</p><p><b>Basic Protocol 8</b>: Whole-genome sequencing using long reads</p><p><b>Basic Protocol 9</b>: De novo genome assembly using the Phage Galaxy platform</p><p><b>Basic Protocol 10</b>: Annotation and characterization of phage genome</p><p><b>Basic Protocol 11</b>: Host range testing and characterization of isolated phages using BLOTS</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"6 9","pages":""},"PeriodicalIF":2.5,"publicationDate":"2026-08-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13528971/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148868175","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}