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Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology. 利用RPA-CRISPR-Cas12a技术检测生态珍稀鱼类eDNA的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-10 DOI: 10.1016/j.xpro.2024.103544
Xing-Yi Wei, Yang Pei, Li Liu, Péter Hamar, De-Sheng Pei
{"title":"Protocol for detecting eDNA in ecological rare fish using RPA-CRISPR-Cas12a technology.","authors":"Xing-Yi Wei, Yang Pei, Li Liu, Péter Hamar, De-Sheng Pei","doi":"10.1016/j.xpro.2024.103544","DOIUrl":"10.1016/j.xpro.2024.103544","url":null,"abstract":"<p><p>The recombinase polymerase amplification (RPA)-CRISPR-Cas12a-FQ system enables sensitive detection of environmental DNA (eDNA) in rare fish species. Here, we present a protocol for eDNA amplification and Cas12a for target recognition using RPA. We describe steps for identifying a target site, synthesis and purification of CRISPR RNA (crRNA), and RPA isothermal amplification. We then detail procedures for constructing the eDNA CRISPR-Cas12a detection system and verifying its sensitivity. This protocol offers a high-sensitivity approach for monitoring aquatic biodiversity and conservation efforts, even in low eDNA concentrations. For complete details on the use and execution of this protocol, please refer to Wei et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103544"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11772130/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972427","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}
引用次数: 0
Protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique. 使用crispr - cas9介导的敲入技术生成斑马鱼周细胞报告细胞系Ki（pdgfrb-P2A-GAL4-VP16）的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2024-12-13 DOI: 10.1016/j.xpro.2024.103490
Huaxing Zi, Xiaolan Peng, Jiulin Du, Jia Li
{"title":"Protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique.","authors":"Huaxing Zi, Xiaolan Peng, Jiulin Du, Jia Li","doi":"10.1016/j.xpro.2024.103490","DOIUrl":"10.1016/j.xpro.2024.103490","url":null,"abstract":"<p><p>Pericytes, the mural cells that envelop small blood vessels, play crucial roles in the formation of the blood-brain barrier (BBB). Here, we present a protocol for generating a pericyte reporter zebrafish line Ki(pdgfrb-P2A-GAL4-VP16) using a CRISPR-Cas9-mediated knockin technique. We describe steps for identifying efficient single guide RNA (sgRNA), constructing donor plasmid, and generating and maintaining the knockin line. We then detail procedures for in vivo imaging of brain pericytes. This protocol is adaptable for creating other knockin lines for specific cell labeling. For complete details on the use and execution of this protocol, please refer to Zi et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103490"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11699729/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142824640","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}
引用次数: 0
Protocol to compare relative protein-liposome binding affinity using a fluorescence microscopy-based approach. 使用基于荧光显微镜的方法比较相对蛋白-脂质体结合亲和力的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2024-12-14 DOI: 10.1016/j.xpro.2024.103507
Kar Ling Hoh, Dan Zhang
{"title":"Protocol to compare relative protein-liposome binding affinity using a fluorescence microscopy-based approach.","authors":"Kar Ling Hoh, Dan Zhang","doi":"10.1016/j.xpro.2024.103507","DOIUrl":"10.1016/j.xpro.2024.103507","url":null,"abstract":"<p><p>Centrifugation-based protein-liposome assays are unsuitable for spontaneously precipitating proteins and have limited quantification capabilities. Here, we present a protocol to compare relative protein-liposome binding affinity using a fluorescence microscopy-based approach. We described steps for fluorescent liposome preparation, fission yeast protein extraction, liposome binding assay, and confocal imaging. We also provided analysis methods for different setups. Although this protocol is established for fission yeast, it can be applied to most non-transmembrane proteins obtained from various in vivo and in vitro systems. For complete details on the use and execution of this protocol, please refer to Hoh et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103507"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11699414/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142830191","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}
引用次数: 0
Protocol for generating brain metastatic tumor cells through repeated intracardiac injections in mice. 通过小鼠心脏内反复注射产生脑转移性肿瘤细胞的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2024-12-20 DOI: 10.1016/j.xpro.2024.103531
Li Wang, Xiaoxia Chen, Chengtao Dong, Shengqi Yin, Li Liang, Aidong Zhou
{"title":"Protocol for generating brain metastatic tumor cells through repeated intracardiac injections in mice.","authors":"Li Wang, Xiaoxia Chen, Chengtao Dong, Shengqi Yin, Li Liang, Aidong Zhou","doi":"10.1016/j.xpro.2024.103531","DOIUrl":"10.1016/j.xpro.2024.103531","url":null,"abstract":"<p><p>Here, we present a protocol for generating the lung cancer cell line, LLC1-BMT5, with highly brain metastatic tropism through multiple rounds of in vivo selection. We describe steps for establishing the brain metastases (BrMs) mouse model through intracardiac injection of cancer cells. We then detail procedures for obtaining brain metastatic subpopulations, in vivo selection of LLC1-BMT5 cells, and validating metastatic potential. This protocol will facilitate the study of the molecular mechanisms of BrMs and the development of anti-BrM treatment strategies. For complete details on the use and execution of this protocol, please refer to Wang et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103531"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11726779/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142872848","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}
引用次数: 0
Protocol for the assessment, improvement, and harmonization of somatic variant calling using ONCOLINER. 使用ONCOLINER评估、改进和协调体细胞变异呼叫的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2024-12-20 DOI: 10.1016/j.xpro.2024.103533
Rodrigo Martín, Nicolás Gaitán, David Torrents
{"title":"Protocol for the assessment, improvement, and harmonization of somatic variant calling using ONCOLINER.","authors":"Rodrigo Martín, Nicolás Gaitán, David Torrents","doi":"10.1016/j.xpro.2024.103533","DOIUrl":"10.1016/j.xpro.2024.103533","url":null,"abstract":"<p><p>The interoperability of variant identification pipelines is fundamental for achieving consistent clinical care across oncology research centers and hospitals. Here, we present a protocol for using ONCOLINER, a platform for the assessment, improvement, and harmonization of somatic variant discovery of multiple pipelines. We describe steps for acquiring benchmarking datasets and executing the user variant calling pipeline. We then detail the procedures for performing analyses to produce user-friendly reports showing the quality, scope, and applicable improvements for each tumor genome analysis. For complete details on the use and execution of this protocol, please refer to Martín et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103533"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11731501/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142872908","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}
引用次数: 0
Protocol for investigating intracellular microbial diversity using single-cell RNA-seq in immune cells of SARS-CoV-2-positive and recovered patients. 利用单细胞RNA-seq技术研究sars - cov -2阳性和康复患者免疫细胞内微生物多样性的方案
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-08 DOI: 10.1016/j.xpro.2024.103546
Jyoti Soni, Priyanka Mehta, Sunita Yadav, Partha Chattopadhyay, Rajesh Pandey
{"title":"Protocol for investigating intracellular microbial diversity using single-cell RNA-seq in immune cells of SARS-CoV-2-positive and recovered patients.","authors":"Jyoti Soni, Priyanka Mehta, Sunita Yadav, Partha Chattopadhyay, Rajesh Pandey","doi":"10.1016/j.xpro.2024.103546","DOIUrl":"10.1016/j.xpro.2024.103546","url":null,"abstract":"<p><p>Intracellular microorganisms like viruses and bacteria impact immune cell function. However, detection of these microbes is challenging as the majority exist in a non-culturable state. This protocol presents detailed steps to investigate intracellular microbial diversity using single-cell RNA sequencing (scRNA-seq) in immune-cells of SARS-CoV-2-positive and recovered patients. We present a workflow from sample collection to library preparation, covering peripheral blood mononuclear cell (PBMC) isolation, single-cell labeling, cartridge priming, and cell lysis. We outline the steps for analyzing the scRNA-seq data, from data quality control (QC) to detection of intracellular microbes. For complete details on the use and execution of this protocol, please refer to Yadav et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103546"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11780099/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142956075","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}
引用次数: 0
Protocol for recombinant rabies virus titration by quantitative PCR.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-22 DOI: 10.1016/j.xpro.2024.103567
He Zhang, Xueping Gao, Xiangyu Ge, Xiao Wang, Minghui Song, Xia Zhang, Lei Jin
{"title":"Protocol for recombinant rabies virus titration by quantitative PCR.","authors":"He Zhang, Xueping Gao, Xiangyu Ge, Xiao Wang, Minghui Song, Xia Zhang, Lei Jin","doi":"10.1016/j.xpro.2024.103567","DOIUrl":"10.1016/j.xpro.2024.103567","url":null,"abstract":"<p><p>Preparing high-titer virus and performing accurate titer determination are critical to subsequent experiments. However, not all applied recombinant rabies viruses, such as the L-deleted virus,<sup>1</sup> are equipped with fluorescent proteins for titration by fluorescence-activated cell sorting (FACS). Here, we present a quantitative reverse-transcription PCR (RT-qPCR) approach for titrating recombinant rabies virus. We describe steps for preparing standards for RT-qPCR, rabies virus genome RNA extraction, and reverse transcription of virus RNA. We then detail procedures for RT-qPCR for titration and stereotaxic rabies virus injection for titer verification.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103567"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11794156/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143029622","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}
引用次数: 0
Optimized protocol for single-cell isolation and alkaline comet assay to detect DNA damage in cells of Drosophila wing imaginal discs.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-23 DOI: 10.1016/j.xpro.2024.103590
Melissa Pederzolli, Edoardo Barion, Anna Valerio, Andrea Vutera Cuda, Valeria Manara, Paola Bellosta
{"title":"Optimized protocol for single-cell isolation and alkaline comet assay to detect DNA damage in cells of Drosophila wing imaginal discs.","authors":"Melissa Pederzolli, Edoardo Barion, Anna Valerio, Andrea Vutera Cuda, Valeria Manara, Paola Bellosta","doi":"10.1016/j.xpro.2024.103590","DOIUrl":"10.1016/j.xpro.2024.103590","url":null,"abstract":"<p><p>Reduced expression of nucleolar genes induces stress and DNA damage. Here, we present a protocol to analyze DNA fragmentation at the single-cell level in Drosophila imaginal discs using an optimized alkaline comet assay. We describe steps for larvae development, tissue disaggregation, and single-cell dissociation. We then detail procedures for cell lysis, electrophoresis, and DNA visualization. This approach provides insights into the molecular consequences of nucleolar stress induction and its role in the DNA damage response pathways in vivo.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103590"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11804111/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143034441","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}
引用次数: 0
Protocol for real-time assessment of energy metabolism in dissociated mouse retinal photoreceptors using a SeahorseXFe24 analyzer.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-23 DOI: 10.1016/j.xpro.2025.103595
Maria Yera, Joshua J Wang, Sarah X Zhang
{"title":"Protocol for real-time assessment of energy metabolism in dissociated mouse retinal photoreceptors using a SeahorseXFe24 analyzer.","authors":"Maria Yera, Joshua J Wang, Sarah X Zhang","doi":"10.1016/j.xpro.2025.103595","DOIUrl":"10.1016/j.xpro.2025.103595","url":null,"abstract":"<p><p>Defects in retinal metabolism have been linked to the onset and progression of various retinal diseases. Herein, we provide a protocol for measuring bioenergetics in dissociated mouse retinal photoreceptors. We outline detailed instructions for obtaining morphologically intact and viable photoreceptor cells from adult mice and preparing the cells for metabolic analysis using a SeahorseXFe24 analyzer. This protocol allows a real-time assessment of mitochondrial respiration and glycolysis in retinal photoreceptors in response to genetic modifications or pathological insults in mouse models.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103595"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11795547/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143034448","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}
引用次数: 0
Computational protocol for analyzing whole-genome sequencing data from Staphylococcus aureus clinical isolates.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-31 DOI: 10.1016/j.xpro.2025.103613
Miquel Sánchez-Osuna, Ivan Erill, Oriol Gasch, Oscar Q Pich
{"title":"Computational protocol for analyzing whole-genome sequencing data from Staphylococcus aureus clinical isolates.","authors":"Miquel Sánchez-Osuna, Ivan Erill, Oriol Gasch, Oscar Q Pich","doi":"10.1016/j.xpro.2025.103613","DOIUrl":"10.1016/j.xpro.2025.103613","url":null,"abstract":"<p><p>Analyzing whole-genome sequencing (WGS) data from bacterial isolates is pivotal for understanding virulence and predicting clinical outcomes through association studies. Herein, we present a computational protocol for the detailed analysis of WGS data from Staphylococcus aureus clinical isolates generated with Illumina sequencing. We describe steps for de novo assembly, functional annotation, and genetic characterization of chromosomal and extrachromosomal elements. This approach paves the way for an improved understanding of the interplay between virulence factors, resistome, strain type, and disease severity. For complete details on the use and execution of this protocol, please refer to Sánchez-Osuna et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103613"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11835644/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143075701","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}
引用次数: 0
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