Bio-protocol最新文献

{"title":"Precise Generation of Human Induced Pluripotent Stem Cell-derived Cell Lines Harboring Disease-relevant Single Nucleotide Variants Using a Prime Editing System.","authors":"Seiya Kanno, Kota Sato, Toru Nakazawa","doi":"10.21769/BioProtoc.5191","DOIUrl":"10.21769/BioProtoc.5191","url":null,"abstract":"<p><p>Human induced pluripotent stem (iPS) cell lines harboring mutations in disease-related genes serve as invaluable in vitro models for unraveling disease mechanisms and accelerating drug discovery efforts. Introducing mutations into iPS cells using traditional gene editing approaches based on the CRISPR-Cas9 endonuclease often encounters challenges such as unintended insertions/deletions (indels) and off-target effects. To address these limitations, we present a streamlined protocol for introducing highly accurate gene mutations into human iPS cells using prime editing, a \"search-and-replace\" genome-editing technology that combines unwanted indel-minimized CRISPR-Cas9 nickase with reverse transcriptase. This protocol encompasses the design of prime editing guide RNAs (pegRNAs) required for binding and replacement at target loci, construction of prime editor and pegRNA expression vectors, gene transfer into iPS cells, and cell line selection. This protocol allows for the efficient establishment of disease-associated gene variants within 6-8 weeks while preserving critical genomic context. Key features • Dramatic improvement in efficiency of In-Fusion cloning using inserts assembled from the three pegRNA components (spacer, spCas9 scaffold, and 3' extension) via overlap extension PCR. • Cost-effective and time-saving selection of pegRNAs for prime editing via bulk Sanger sequencing. • Straightforward gene transfection using polymer-based reagents, which requires no specialized equipment or techniques and offers high reproducibility and broad applicability across different cell lines. • Precise genome editing based on pegRNA/prime editing minimizes off-target effects, enabling a wide range of applications in the study of disease-associated genetic variants. Graphical overview Key steps of generation of human induced pluripotent stem (iPS) cell lines harboring disease-relevant single nucleotide variants (SNVs) using a prime editing system.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5191"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865833/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545197","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":"Visualization of Gap Junction-Mediated Astrocyte Coupling in Acute Mouse Brain Slices.","authors":"Nine F Kompier, Gabrielle Siemonsmeier, Niklas Meyer, Helmut Kettenmann, Fritz G Rathjen","doi":"10.21769/BioProtoc.5220","DOIUrl":"https://doi.org/10.21769/BioProtoc.5220","url":null,"abstract":"<p><p>Gap junctions are transmembrane protein channels that enable the exchange of small molecules such as ions, second messengers, and metabolites between adjacent cells. Gap junctions are found in various mammalian organs, including skin, endothelium, liver, pancreas, muscle, and central nervous system (CNS). In the CNS, they mediate coupling between neural cells including glial cells, and the resulting panglial networks are vital for brain homeostasis. Tracers of sufficiently small molecular mass can diffuse across gap junctions and are used to visualize the extent of cell-to-cell coupling in situ by delivering them to a single cell through sharp electrodes or patch-clamp micropipettes. Here, we describe a protocol for pre-labeling and identification of astrocytes in acute mouse forebrain slices using Sulforhodamine 101 (SR101). Fluorescent cells can then be targeted for whole-cell patch-clamp, which allows for further confirmation of astroglial identity by assessing their electrophysiological properties, as well as for passive dialysis with a tracer such as biocytin. Slices can then be subjected to chemical fixation and immunostaining to detect dye-coupled networks. This protocol provides a method for the identification of astrocytes in live tissue through SR101 labeling. Alternatively, transgenic reporter mice can also be used to identify astrocytes. While we illustrate the use of this protocol for the study of glial networks in the mouse brain, the general principles are applicable to other species, tissues, and cell types. Key features • Pre-labeling of live astrocytes in acute adult mouse brain slices using the dye Sulforhodamine 101. • Dialysis of biocytin into individual astrocytes using whole-cell patch-clamp electrophysiology. • Staining of biocytin by streptavidin and immunostaining of GFAP, imaging, and analysis of dye-coupled astrocytic networks. • Can be used for other glial cell types and might be adapted to other tissues and species.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5220"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865822/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143544813","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":"Vegetative Propagation of <i>Cannabis sativa</i> and Resin Obtained From its Female Inflorescences.","authors":"Sebastián D Ippolito, Marina Landaburu, María E Vozza Berardo, María D Villamonte, Julieta R Mendieta, Débora Nercessian, Silvana L Colman","doi":"10.21769/BioProtoc.5204","DOIUrl":"https://doi.org/10.21769/BioProtoc.5204","url":null,"abstract":"<p><p>Cannabis (<i>Cannabis sativa</i> L.) derivatives are of great importance in the medical, cosmetic, and pharmaceutical industries. This relevance is mainly due to the active principles (cannabinoids) found mainly in the trichomes of the female inflorescences. One of the most commonly used methods to propagate cannabis is by vegetative stem cuttings. This low-cost technique produces genetically uniform plants, ensuring consistent growth rates and cannabinoid production. The extraction of cannabinoids and other active compounds from the resin of the flowers is the main limitation of cannabis processing. Here, we describe a step-by-step protocol for propagating female cannabis plants from vegetative stem cuttings, inducing flower development, and obtaining high-quality cannabinoid-enriched resin. Key features • The propagation of cannabis is done by vegetative cuttings. • It takes 4-5 months to cultivate the plant and obtain female inflorescences. • Resin is the end product of the alcoholic extraction of flowers, and the obtained resin is a full-spectrum mixture of active compounds.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5204"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865818/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143544336","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":"Nuclei Isolation From Murine and Human Periosteum For Transcriptomic Analyses.","authors":"Simon Perrin, Cassandre Goachet, Maria Ethel, Yasmine Hachemi, Céline Colnot","doi":"10.21769/BioProtoc.5211","DOIUrl":"10.21769/BioProtoc.5211","url":null,"abstract":"<p><p>Bone repair is a complex regenerative process relying on skeletal stem/progenitor cells (SSPCs) recruited predominantly from the periosteum. Activation and differentiation of periosteal SSPCs occur in a heterogeneous environment, raising the need for single cell/nucleus transcriptomics to decipher the response of the periosteum to injury. Enzymatic cell dissociation can induce a stress response affecting the transcriptome and lead to overrepresentation of certain cell types (i.e., immune and endothelial cells) and low coverage of other cell types of interest. To counteract these limitations, we optimized a protocol to isolate nuclei directly from the intact periosteum and from the fracture callus to perform single-nucleus RNA sequencing. This protocol is adapted for fresh murine periosteum, fracture callus, and frozen human periosteum. Nuclei are isolated using mechanical extraction combined with fluorescence-based nuclei sorting to obtain high-quality nucleus suspensions. This protocol allows the capture of the full diversity of cell types in the periosteum and fracture environment to better reflect the in vivo tissue composition. Key features • Allows the isolation of nuclei with high-quality RNA for transcriptomic analyses. • Can be adapted to be used on fresh and frozen tissue. • Optimized for human and murine periosteum.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5211"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865820/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545196","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":"Combined FLIM, Confocal Microscopy, and STED Nanoscopy for Live-Cell Imaging.","authors":"Magalie Bénard, Christophe Chamot, Damien Schapman, Alexis Lebon, Ludovic Galas","doi":"10.21769/BioProtoc.5202","DOIUrl":"https://doi.org/10.21769/BioProtoc.5202","url":null,"abstract":"<p><p>Time-lapse fluorescence microscopy is a relevant technique to visualize biological events in living samples. Maintaining cell survival by limiting light-induced cellular stress is challenging and requires protocol development and image acquisition optimization. Here, we provide a guide by considering the quartet <i>sample, probe, instrument</i>, and <i>image processing</i> to obtain appropriate resolutions and information for live cell fluorescence imaging. The pleural mesothelial cell line H28, an adherent cell line that is easy to seed, was used to develop innovative advanced light microscopy strategies. The chosen red and near-infrared probes, capable of passively penetrating through the cell plasma membrane, are particularly suitable because their stimulation from 600 to 800 nm induces less cytotoxicity. The labeling protocol describes the concentration, time, and incubation conditions of the probes and associated adjustments for multi-labeling. To limit phototoxicity, acquisition parameters in advanced confocal laser scanning microscopy with a white laser are determined. Light power must be adjusted and minimized at red wavelengths for reduced irradiance (including a 775 nm depletion laser for STED nanoscopy), in simultaneous mode with hybrid detectors and combined with the fast FLIM module. These excellent conditions allow us to follow cellular and intracellular dynamics for a few minutes to several hours while maintaining good spatial and temporal resolutions. Lifetime analysis in lifetime imaging (modification of the lifetime depending on environmental conditions), lifetime dye unmixing (separation with respect to the lifetime value for the spectrally closed dye), and lifetime denoising (improvement of image quality) provide flexibility for multiplexing experiments. Key features • Cell preservation after labeling with less cytotoxic red, near-infrared dye viable probes. • Determination of lower but efficient probe concentration; adjust good balance between probes concentration and incubation time to achieve multi-labeling. • Long time-lapse acquisition in advanced confocal microscopy with sensitive new-generation detectors. • Confocal image combined with fast FLIM for multi-labeling with spectrally closed dyes, unmixed from lifetime values. • Confocal-STED image acquisition combined with fast FLIM to improve signal-to-noise ratio.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 4","pages":"e5202"},"PeriodicalIF":1.0,"publicationDate":"2025-02-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11865824/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143545177","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":"Rapid Sampling of Large Quantities of Interstitial Fluid from Human Skin Using Microneedles and a Vacuum-assisted Skin Patch.","authors":"Elizabeth C Wilkirson, Xue Jiang, Peter B Lillehoj","doi":"10.21769/BioProtoc.5173","DOIUrl":"10.21769/BioProtoc.5173","url":null,"abstract":"<p><p>Interstitial fluid (ISF) is a promising diagnostic sample due to its extensive biomolecular content while being safer and less invasive to collect than blood. However, existing ISF sampling methods are time-consuming, require specialized equipment, and yield small amounts of fluid (<5 μL). We have recently reported a simple and minimally invasive technique for rapidly sampling larger quantities of dermal ISF using a microneedle (MN) array to generate micropores in the skin from which ISF is extracted using a vacuum-assisted skin patch. Here, we present step-by-step protocols for fabricating the MN array and skin patch, as well as for using them to sample ISF from human skin. Using this technique, an average of 20.8 μL of dermal ISF can be collected within 25 min, which is a ∼6-fold improvement over existing ISF sampling methods. Furthermore, the technique is well-tolerated and does not require the use of expensive or specialized equipment. The ability to collect ample volumes of ISF in a quick and minimally invasive manner will facilitate the analysis of ISF for biomarker discovery and its use for diagnostic testing. Key features • Minimally invasive (bloodless and nearly painless) technique for sampling ISF from human skin. • An average of 20.8 μL of interstitial fluid can be collected within 25 min. • This technique does not require expensive or specialized equipment or electricity. • Collected ISF can be analyzed using conventional laboratory-based assays or point-of-care diagnostic tests.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5173"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825300/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434651","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":"Optimal Dual RNA-Seq Mapping for Accurate Pathogen Detection in Complex Eukaryotic Hosts.","authors":"Infanta Saleth Teresa Eden M, Umashankar Vetrivel","doi":"10.21769/BioProtoc.5182","DOIUrl":"10.21769/BioProtoc.5182","url":null,"abstract":"<p><p>Dual RNA-Seq technology has significantly advanced the study of biological interactions between two organisms by allowing parallel transcriptomic analysis. Existing analysis methods employ various combinations of open-source bioinformatics tools to process dual RNA-Seq data. Upon reviewing these methods, we intend to explore crucial criteria for selecting standard tools and methods, especially focusing on critical steps such as trimming and mapping reads to the reference genome. In order to validate the different combinatorial approaches, we performed benchmarking using top-ranking tools and a publicly available dual RNA-Seq Sequence Read Archive (SRA) dataset. An important observation while evaluating the mapping approach is that when the adapter trimmed reads are first mapped to the pathogen genome, more reads align to the pathogen genome than the unmapped reads derived from the traditional host-first mapping approach. This mapping method prevents the misalignment of pathogen reads to the host genome due to their shorter length. In this way, the pathogenic read information found at lesser proportions in a complex eukaryotic dataset is precisely obtained. This protocol presents a comprehensive comparison of these possible approaches, resulting in a robust unified standard methodology. Key features • Benchmarking of top-ranking software for quality control, adapter trimming, and read mapping. • Emphasizes the importance of read mapping criteria for dual RNA-Seq datasets: (i) high count of uniquely host mapped reads, (ii) low count of host multi-mapped reads, and (iii) high count of unmapped reads belonging to pathogens. • Elaborates the best mapping approach to precisely extract the pathogen reads as these get captured comparatively less in dual RNA-Seq datasets. Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5182"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825298/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434471","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":"Isolation of Intact Mitochondria From <i>Drosophila melanogaster</i> and Assessment of Mitochondrial Respiratory Capacity Using Seahorse Analyzer.","authors":"Christopher M Groen, Anthony J Windebank","doi":"10.21769/BioProtoc.5180","DOIUrl":"10.21769/BioProtoc.5180","url":null,"abstract":"<p><p>Analysis of mitochondrial function has broad applicability in many research specialties. Neurodegenerative disorders such as chemotherapy-induced peripheral neuropathy (CIPN) often exhibit damaged mitochondria or reduced mitochondrial respiratory capacity. Isolation of intact mitochondria for protein analysis or respiration measurements has been previously reported in numerous model organisms. Here, we describe an adaptation of previous protocols to isolate intact functional mitochondria from <i>Drosophila melanogaster</i> for use in a model of CIPN. Whole <i>Drosophila</i> are ground in isolation buffer, and mitochondria are purified using differential centrifugation through a sucrose and mannitol solution. The intact mitochondria are plated as a monolayer for measurements of mitochondrial oxygen consumption rates and response to inhibitor compounds on an Agilent Seahorse analyzer. This experimental protocol is quick and yields a purified population of intact mitochondria that may be used for functional assays for several hours after isolation. The isolated mitochondria may be used for respiration measurements, which reflect their health, and stored for protein or genetic analysis. Mitochondrial populations from multiple strains or treatment groups can be easily compared simultaneously. The rapid biochemical assessment of mitochondria, in combination with the utility of <i>Drosophila</i> as an in vivo genetic model system, offers great potential for researchers to probe the impact of genetics and pharmacologic interventions on mitochondrial respiratory capacity. Key features • This protocol describes rapid isolation of intact, functional mitochondria that may be used for respiration measurements or other biochemical analyses. • Mitochondria isolated from <i>Drosophila</i> are assessed in an Agilent Seahorse analyzer utilizing multiple substrates and electron transport chain inhibitors to fully characterize mitochondrial respiratory capacity. • This protocol is optimized to use <i>Drosophila</i> for easy in vivo genetic and pharmacologic manipulation, and assessment of the impact on mitochondrial function.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5180"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825302/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434016","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":"Development and Validation of <i>Chlamydia muridarum</i> Mouse Models for Studying Genital Tract Infection Pathogenesis.","authors":"Yihui Wang, Zixuan Han, Luying Wang, Xin Sun, Qi Tian, Tianyuan Zhang","doi":"10.21769/BioProtoc.5181","DOIUrl":"10.21769/BioProtoc.5181","url":null,"abstract":"<p><p>Animal infection models play significant roles in the study of bacterial pathogenic mechanisms and host-pathogen interactions, as well as in evaluating drug and vaccine efficacies. <i>Chlamydia trachomatis</i> is responsible for infections in various mucosal tissues, including the eyes and urogenital, respiratory, and gastrointestinal tracts. Chronic infections can result in severe consequences such as trachoma-induced blindness, ectopic pregnancy, and infertility. While intravaginal inoculation of <i>C. muridarum</i> mimics the natural route of sexual transmission between individuals, transcervical inoculation allows the organisms to directly infect endometrial epithelial cells without interference from host responses triggered by chlamydial contact or infection of vaginal and cervical cells. Therefore, in this study, we used mouse models to visualize pathologies in both the endometrium and oviduct following <i>C. muridarum</i> inoculation. Key features • This protocol develops the mouse-adapted <i>Chlamydia muridarum</i> model, ideal for visualizing pathologies in both the endometrium and oviduct genital tract. • Requires female mice and utilizes specific techniques for intravaginal and transcervical inoculation with chlamydial elementary body (EB) and a form specialized for intracellular replication. • The protocol necessitates specialized equipment, including a laminar flow hood, a micropipette, and a non-surgical embryo transfer device (NSET). Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5181"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825293/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434524","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":"An Effective and Safe Maize Seed Chipping Protocol Using Clipping Pliers With Applications in Small-Scale Genotyping and Marker-Assisted Breeding.","authors":"Brian Zebosi, John Ssengo, Lander F Geadelmann, Erica Unger-Wallace, Erik Vollbrecht","doi":"10.21769/BioProtoc.5200","DOIUrl":"10.21769/BioProtoc.5200","url":null,"abstract":"<p><p>In applications such as marker-assisted breeding and positional cloning, tissue sampling and plant tracking are vital steps in the genotyping pipeline. They enable the identification of desirable seedlings, saving time and reducing the cost, space, and handling required for growing adult plants, especially for greenhouses and winter nurseries. Small-scale marker-assisted selection laboratories rely heavily on leaf-based genotyping, which involves over-planting large, segregating populations followed by leaf sampling, genotyping, and backtracking to identify desired individuals, which is costly and laborious. Thus, there is a need to adopt seed-based genotyping to reduce costs and save time. Therefore, we developed a safe and cheap seed-chipping protocol using clipping pliers to chip seeds to genotype before planting. To identify a cost-effective and high-throughput DNA extraction method, we tested four extraction methods and assessed the quality of the seed DNA using PCR. For three of the methods, seed-based DNA was of comparable quality to DNA extracted from leaf punches. We also compared seed- and leaf-derived DNA from the same individuals in a segregating population to test for genotyping miscalls that could arise due to the presence of maternally derived pericarp in the seed samples. Out of 43 potential instances, we found zero miscalled samples and, therefore, no evidence supporting consequential pericarp inclusion. Germination rates of chipped and unchipped seeds were the same for the inbreds tested, B73 and Mo17. However, chipped seeds grew slower until ~14 days after sowing. Overall, seed sampling using clipping pliers provides a simple, reliable, and high-throughput method to identify specific genotypes before planting. Key features • Provides a quick, safe, and cheap sampling technique for maize kernels that may also be suitable for other plants with relatively large seeds. • Includes procedures and materials to track and organize samples within and across batches involving tens to thousands of seeds. • Seeds can be sampled and genotyped relatively quickly for planting; in one day, 384 seeds can be sampled, processed for DNA, and genotyped by PCR.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 3","pages":"e5200"},"PeriodicalIF":1.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11825296/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143434592","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}