Bio-protocol最新文献

{"title":"Egg Microinjection for the Silkworm <i>Bombyx mori</i>.","authors":"Hayato Yamada, Toshinobu Yaginuma, Keisuke Mase, Teruyuki Niimi","doi":"10.21769/BioProtoc.5317","DOIUrl":"10.21769/BioProtoc.5317","url":null,"abstract":"<p><p>The silkworm <i>Bombyx mori</i> has been extensively utilized in sericulture and serves as a representative model insect of Lepidoptera in various fields of life sciences and applied research. In recent years, its significance has further increased in molecular genetics and functional genomics. Germline transformation and genome editing in <i>B. mori</i> require the injection of vector solutions into early embryos; however, the thick eggshell of <i>B. mori</i> presents a significant challenge for microinjection. Conventional methods involve arranging eggs, pre-pierced with a tungsten needle, followed by solution injection, making the process both time-consuming and technically demanding. Here, we describe a simplified and more efficient microinjection protocol. Unlike conventional approaches, our method eliminates the need for egg removal from the egg-laying sheet and egg alignment on the slide glass by allowing injections to be performed directly on eggs retained on the egg-laying sheet. A thick-walled glass capillary, capable of penetrating the rigid eggshell, is used to directly pierce the eggshell and deliver the solution. By eliminating the need for egg alignment and micromanipulator operation, this protocol significantly enhances efficiency, enabling higher-throughput embryo injections within a shorter time frame. Moreover, this approach holds potential for application to other insect species with similarly thick eggshells. Key features • A simple and efficient microinjection method for silkworm eggs. • Eliminates the need for egg alignment by injecting directly into eggs attached to the oviposition substrate. • Conducted without a micromanipulator or tungsten needle to pierce the eggshell, using only a handheld thick-walled glass capillary. • Applicable to other insect species with thick eggshells.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5317"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104832/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164005","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":"<i>Mycobacterium smegmatis</i> Ribosome Purification, Co-sedimentation, and Subunit Association Assay.","authors":"Aneek Banerjee, Sandip Dey, Krishnamoorthi Srinivasan, Ankit Dhur, Priya Baid, Jayati Sengupta","doi":"10.21769/BioProtoc.5318","DOIUrl":"10.21769/BioProtoc.5318","url":null,"abstract":"<p><p>The ribosome, a complex macromolecular machine, plays a vital role in cellular translation. To investigate its structure and conduct in vitro experiments, isolating the ribosomes from cells is the first step. While isolating ribosomes from bacterial cells is routine, obtaining them from mycobacteria proves challenging due to the protective mycolic acid layer, which hinders cell lysis. In this study, we present a straightforward and efficient protocol for isolating ribosomes from <i>Mycobacterium smegmatis</i>. Additionally, we introduce a co-sedimentation assay using density gradient ultracentrifugation, providing a simple yet powerful method for studying ribosome-protein interactions. The re-association assay also offers a practical approach for obtaining tRNA-free 70S ribosomes and evaluating the anti-association properties of potential ligands. While these assays are commonly used, our protocol stands out for its simplicity, requiring limited specialized instruments. These methods can also be scaled up or down per requirement. By employing sonication for cell rupture and utilizing basic lab equipment for ultracentrifugation-based assays, our method greatly simplifies ribosome isolation and related research. Key features • Cellular ribosome isolation from <i>Mycobacterium smegmatis</i> using an optimized sonication cycle. • Detection of ribosome-protein interactions through density gradient ultracentrifugation. • Simple steps to obtain tRNA-free 70S ribosomes that are crucial for several biochemical experiments. • Screening of proteins or ligands for their ribosomal anti-association activity.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5318"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12117347/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144175681","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":"From Bedside to Desktop: A Data Protocol for Normative Intracranial EEG and Abnormality Mapping.","authors":"Heather Woodhouse, Sarah J Gascoigne, Gerard Hall, Callum Simpson, Nathan Evans, Gabrielle M Schroeder, Peter N Taylor, Yujiang Wang","doi":"10.21769/BioProtoc.5321","DOIUrl":"10.21769/BioProtoc.5321","url":null,"abstract":"<p><p>Normative mapping is a framework used to map population-level features of health-related variables. It is widely used in neuroscience research, but the literature lacks established protocols in modalities that do not support healthy control measurements, such as intracranial electroencephalograms (icEEG). An icEEG normative map would allow researchers to learn about population-level brain activity and enable the comparison of individual data against these norms to identify abnormalities. Currently, no standardised guide exists for transforming clinical data into a normative, regional icEEG map. Papers often cite different software and numerous articles to summarise the lengthy method, making it laborious for other researchers to understand or apply the process. Our protocol seeks to fill this gap by providing a dataflow guide and key decision points that summarise existing methods. This protocol was heavily used in published works from our own lab (twelve peer-reviewed journal publications). Briefly, we take as input the icEEG recordings and neuroimaging data from people with epilepsy who are undergoing evaluation for resective surgery. As final outputs, we obtain a normative icEEG map, comprising signal properties localised to brain regions. Optionally, we can also process new subjects through the same pipeline and obtain their z-scores (or centiles) in each brain region for abnormality detection and localisation. To date, a single, cohesive dataflow pipeline for generating normative icEEG maps, along with abnormality mapping, has not been created. We envisage that this dataflow guide will not only increase understanding and application of normative mapping methods but will also improve the consistency and quality of studies in the field. Key features • Resultant normative maps can be used to test a broad range of hypotheses in the neuroscience field. • Provides a more detailed walkthrough of the methods in the normative mapping study conducted by Taylor et al. [1] and other related publications [2-12]. • Offers flexibility: readers can tailor the final output by considering key decision points included throughout the protocol. • Involves sub-pipelines, which may be useful to researchers in isolation (i.e., icEEG electrode localisation and/or interictal segment selection).</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5321"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104836/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164095","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":"PBMC-Humanized Mouse Model for Multiple Sclerosis: Studying Immune Changes and CNS Involvement.","authors":"Anastasia Dagkonaki, Irini Papazian, Vasileios Gouzouasis, Ariadni Karles, Maria Kourouvani, Theodore Tselios, Eirini Fragkiadaki, Lesley Probert","doi":"10.21769/BioProtoc.5312","DOIUrl":"10.21769/BioProtoc.5312","url":null,"abstract":"<p><p>Humanized immune system (HIS) mice are powerful tools for studying human immune system function and dysfunction and developing human-specific immunotherapeutics. The availability of sophisticated super immunodeficient mouse strains has allowed immune system humanization using transplants of human peripheral blood mononuclear cells (PBMC) or hematopoietic stem cells. HIS mice are used extensively in immune-oncology, while there are fewer studies in autoimmunity, especially multiple sclerosis (MS). Using the protocol described here, we generated HIS mice that show key features of MS not represented in other widely used MS models [1]. Severely immunodeficient NOD.Cg-<i>B2m^em1Tac Prkdc^scid Il2rg^tm1Sug</i> /JicTac (B2m-NOG) mice, which lack murine B, T, and NK cells and murine major histocompatibility class I molecules and have defective innate immune responses, were transplanted with PBMC from HLA-DRB1-genotyped MS patients and healthy donors. Mice were successfully engrafted with hCD4 and hCD8 T and B lymphocytes and developed both spontaneous and experimental autoimmune encephalomyelitis (EAE)-enhanced T-cell lesions in the central nervous system. B-cell engraftment was highest in mice receiving cells from MS patients with serological evidence for Epstein-Barr virus (EBV) reactivation. This humanized MS model shows advantages over EAE, particularly spontaneous hCD8 T-cell lesions in the brain and spinal cord, mixed hCD8/hCD4 T-cell lesions in EAE-immunized mice, and more severe lesions in mice engrafted with PBMC from MS donors carrying the DRB1*15 MS susceptibility allele compared to DRB1*15-positive healthy and DRB1*13-positive MS donors. MS HIS mice represent simple and rapid tools for investigating human immunopathology and the efficacy of therapeutics at a personalized level. Key features • Humanization of severely immunodeficient B2m-NOG mice with human PBMC. • Engraftment analysis of human immune system in mice using multicolor flow cytometry. • Animal familiarization and handling techniques. • Epstein-Barr virus latency evaluation in human plasma. • Ex vivo characterization of engrafted T-cell cytokine responses.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5312"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104831/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164104","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":"Tracking Oral Nanoparticle Uptake in Mouse Gastrointestinal Tract by Fluorescent Labeling and t-SNE Flow Cytometry.","authors":"Rabeya Jafrin Mow, Michal Pawel Kuczma, Xiaodi Shi, Didier Merlin, Chunhua Yang","doi":"10.21769/BioProtoc.5309","DOIUrl":"10.21769/BioProtoc.5309","url":null,"abstract":"<p><p>The growing demand for advanced analytical techniques to explore complex cellular targets of nanotherapeutics has driven the development of innovative methodologies. This protocol presents a refined approach for fluorescent labeling and flow cytometric analysis of colonic cells following oral lipid nanoparticle (LNP) treatment, focusing on LNP uptake in colonic cell subpopulations in a DSS-induced colitis mouse model. By integrating optimized fluorochrome selection and gating strategies with advanced t-distributed stochastic neighbor embedding (t-SNE) analysis, this method enables precise identification and multidimensional visualization of LNP-targeted epithelial and macrophage populations under the complex conditions of inflamed colon tissue. Building on our previous studies demonstrating the effectiveness of nanoparticles in targeted drug delivery, this approach highlights the utility of flow cytometry for assessing uptake efficiency and cellular targeting. Unlike conventional protocols, it incorporates t-SNE for enhanced multidimensional analysis, allowing for the detection of subtle cellular patterns and the delineation of intricate clusters. By addressing gaps in traditional methodologies, this protocol provides a robust and reproducible framework for investigating in vivo cellular targets and optimizing drug delivery strategies for nanomedicines. Key features • This protocol is optimized for investigating nanoparticle uptake in inflamed colonic tissues from DSS-induced colitis models. • This protocol integrates flow cytometry with t-SNE for high-dimensional data analysis, enabling detailed characterization of cellular populations.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5309"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104837/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164289","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":"Electrophysiological Evaluation of a Sciatic Nerve Degree III Injury Model in Rats.","authors":"Linyu Chen, Ziyi Wang, Chengyao Wang, Ning Ma, Weiqin Zhao, Shuang Liu, Jiajun Lu, Boyuan Zhao, Hong Sun, Pengcheng Che, Na Dou","doi":"10.21769/BioProtoc.5311","DOIUrl":"10.21769/BioProtoc.5311","url":null,"abstract":"<p><p>Sciatic nerve injury is a prevalent traumatic condition that significantly impacts a patient's quality of life. The sciatic nerve compression injury model is among the most commonly utilized models for investigating nerve repair and regeneration. Within this context, the degree III sciatic nerve injury model is frequently employed in scientific research due to its clinical relevance and its suitability for studies focused on functional recovery. However, a standardized approach for accurately assessing the success of constructing the degree III sciatic nerve injury model remains lacking. Traditional macroscopic observation methods exhibit limitations, whereas neurophysiological testing serves as a highly sensitive and objective evaluation technique that can directly reflect changes in nerve conduction function, thus providing reliable quantitative evidence for the successful establishment of the model. This study aims to offer a comprehensive description of the application of neurophysiological techniques in evaluating the construction of the degree III sciatic nerve injury model, thereby ensuring the success of model preparation. Key features • Precisely simulate a degree III sciatic nerve injury. • Conduct neurophysiological tests on the sciatic nerve 30 min after the injury. • Use neurophysiological techniques to improve the accuracy and repeatability of scientific study results.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5311"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104839/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164013","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":"Synchronized Visualization and Analysis of Intracellular Trafficking and Maturation of <i>Orthoflavivirus</i> Subviral Particles.","authors":"Kotaro Ishida, Eiji Morita","doi":"10.21769/BioProtoc.5324","DOIUrl":"10.21769/BioProtoc.5324","url":null,"abstract":"<p><p><i>Orthoflavivirus</i> is an enveloped, positive-stranded RNA virus that buds into the endoplasmic reticulum (ER) lumen. The budded virus particles are subsequently transported to the Golgi apparatus and secreted into the extracellular environment via the conventional secretion pathway. In this protocol, we describe a method for monitoring the secretion of <i>Orthoflavivirus</i> particles from the ER. To visualize intracellular membrane trafficking, we combine two distinct imaging techniques: the retention using selective hooks (RUSH) system and the split green fluorescent protein (GFP) system. In this approach, GFP11, a peptide tag fused to prME, the outer coat structural protein of Japanese encephalitis virus particles, was co-expressed in HeLa cells along with two additional components: GFP1-10 fused to a streptavidin-binding peptide and a <i>hook</i> construct consisting of streptavidin fused to the ER retention sequence KDEL. Time-lapse imaging was performed after the addition of biotin, which releases the captured GFP-labeled subviral particles from the ER. This method enables synchronized visualization of intracellular subviral particle trafficking and serves as a valuable tool for analyzing the maturation process of <i>Orthoflavivirus</i> particles within cells. Key features • Synchronized intracellular movement of <i>Orthoflavivirus</i> particles is visualized by the retention using selective hooks (RUSH) system. • Split GFP system is used to label viral particles. • This protocol has broader applications in investigating the transport of secretory proteins, especially those that are challenging to tag with full-length fluorescent proteins.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5324"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104876/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164285","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 Miniprep of Intact Chloroplasts from <i>Arabidopsis thaliana</i> Leaves.","authors":"Brenda A Carranza-Correa, Karen M Dahlman-Cabrera, Manuel Gutiérrez-Aguilar","doi":"10.21769/BioProtoc.5313","DOIUrl":"10.21769/BioProtoc.5313","url":null,"abstract":"<p><p>Cell subfractionation is a common technique employed in many research laboratories to isolate organelles or intracellular compartments for the study of metabolism or biomolecule purification. While numerous protocols exist for isolating organelles, few are specifically designed for starting materials in the milligram range. Here, we present a detailed milligram-scale miniprep protocol for purifying intact chloroplasts from <i>Arabidopsis thaliana</i> leaves. This chloroplast miniprep procedure is suitable for applications such as confocal microscopy, western blotting, enzymatic assays, and other downstream analyses. Key features • This protocol condenses key aspects of existing methods and adapts them to the miniprep scale. • Researchers can isolate purified chloroplasts within 1.5 h. • This protocol requires standard laboratory equipment, such as microcentrifuges (ultracentrifuges are not required). Graphical overview.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5313"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104829/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164158","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":"Dissection and Whole-Mount Immunofluorescent Staining of Mouse Hind Paw Muscles for Neuromuscular Junction Analysis.","authors":"Rebecca L Simkin, Elena R Rhymes, Qiuhan Lang, Nicol Birsa, James N Sleigh","doi":"10.21769/BioProtoc.5315","DOIUrl":"10.21769/BioProtoc.5315","url":null,"abstract":"<p><p>The neuromuscular junction (NMJ) is a peripheral synaptic connection between a lower motor neuron and skeletal muscle fibre that enables muscle contraction in response to neuronal stimulation. NMJ dysfunction and morphological abnormalities are commonly observed in neurological conditions, including amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and spinal muscular atrophy. Employing precise and reproducible techniques to visualise NMJs in mouse models of neuromuscular disorders is crucial for uncovering aspects of neuropathology, revealing disease mechanisms, and evaluating therapeutic approaches. Here, we present a method for dissecting the deep lumbrical and flexor digitorum brevis (FDB) muscles of the mouse hind paw and describe the process of whole-mount immunofluorescent staining for morphological analysis of NMJs. Similar whole-mount techniques have been applied to other muscles, such as the diaphragm; however, dense connective tissue in adult samples often impedes antibody penetration. Moreover, large hind limb muscles, including the gastrocnemius and tibialis anterior, are commonly used to examine NMJs but require embedding and cryosectioning. These additional steps increase the complexity and duration of the protocol and can introduce sectioning artefacts, including transection of NMJs and disruption of morphology. Using small hind paw muscles enables whole-mounting, which completely eliminates the requirement for embedding and cryosectioning. As a result, the entire neuromuscular innervation pattern can be visualised, allowing a more accurate assessment of NMJ development, denervation, and regeneration in mouse models of neurological disease and nerve injury, which can be applied across all postnatal ages. Key features • Small muscles of the mouse hind paw, i.e., lumbrical and FDB muscles, can be rapidly dissected for whole-mount immunofluorescent analysis without the need for cryosectioning. • This protocol allows visualisation of the entire neuromuscular innervation pattern using axonal (anti-tubulin βIII), pre-synaptic (anti-synaptophysin), and post-synaptic (α-bungarotoxin) markers. • Whole-mount immunofluorescence of hind paw muscles enables assessment of developmental, degenerative, and regenerative phenotypes in young and adult mice across disease and injury models. • High-throughput analysis can be performed using NMJ-Analyser or NMJ-morph to evaluate diverse morphological features of the NMJ.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 10","pages":"e5315"},"PeriodicalIF":1.0,"publicationDate":"2025-05-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12104838/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144164072","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":"A Guide to Basic RNA Sequencing Data Processing and Transcriptomic Analysis.","authors":"Rowayna Shouib, Gary Eitzen, Rineke Steenbergen","doi":"10.21769/BioProtoc.5295","DOIUrl":"10.21769/BioProtoc.5295","url":null,"abstract":"<p><p>RNA sequencing (RNA-Seq) has transformed transcriptomic research, enabling researchers to perform large-scale inspection of mRNA levels in living cells. With the growing applicability of this technique to many scientific investigations, the analysis of next-generation sequencing (NGS) data becomes an important yet challenging task, especially for researchers without a bioinformatics background. This protocol offers a beginner-friendly step-by-step guide to analyze NGS data (starting from raw .fastq files), providing the required codes with an explanation of the different steps and software used. We outline a computational workflow that includes quality control, trimming of reads, read alignment to the genome, and gene quantification, ultimately enabling researchers to identify differentially expressed genes and gain insights on mRNA levels. Multiple approaches to visualize this data using statistical and graphical tools in R are also described, allowing the generation of heatmaps and volcano plots to represent genes and gene sets of interest. Key features • Provides a beginner-friendly protocol for RNA-Seq analysis to obtain insights into gene expression. • Pipeline starts with raw .fastq files and involves analysis in command line/terminal and R (via RStudio). • Yields a variety of output files that represent mRNA levels amongst different samples. Output files include count files, heatmaps, ordered lists of DEGs, and volcano plots.</p>","PeriodicalId":93907,"journal":{"name":"Bio-protocol","volume":"15 9","pages":"e5295"},"PeriodicalIF":1.0,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12067304/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144014263","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}