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Protocol for generating protein profiles and distance-based network analysis of murine tissue slices. 生成蛋白质谱和基于距离的小鼠组织切片网络分析协议。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-20 DOI: 10.1016/j.xpro.2024.103578
Luisa Schmidt, Philipp Antczak, Marcus Krüger
{"title":"Protocol for generating protein profiles and distance-based network analysis of murine tissue slices.","authors":"Luisa Schmidt, Philipp Antczak, Marcus Krüger","doi":"10.1016/j.xpro.2024.103578","DOIUrl":"10.1016/j.xpro.2024.103578","url":null,"abstract":"<p><p>We introduce a protocol for spatial proteomics using thin cryotome sections of mouse skeletal muscle tissue. We describe steps for preparing muscle sections and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses to generate spatial protein profiles along the longitudinal skeletal muscle axis. We detail procedures for scanning longitudinal protein profiles and replacing missing data using a sliding window approach. This protocol has potential for applications in spatial proteomics to other tissues with asymmetric patterns such as the brain and heart tissue. For complete details on the use and execution of this protocol, please refer to Schmidt et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103578"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11831136/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143013062","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 reconstructing ancestral genomes from present-day samples by applying local ancestry inference. 应用本地祖先推断从现今样本重建祖先基因组的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-20 DOI: 10.1016/j.xpro.2024.103580
Xiaoxi Zhang, Baonan Wang, Jia Wen, Yang Gao, Yuwen Pan, Shuhua Xu
{"title":"Protocol for reconstructing ancestral genomes from present-day samples by applying local ancestry inference.","authors":"Xiaoxi Zhang, Baonan Wang, Jia Wen, Yang Gao, Yuwen Pan, Shuhua Xu","doi":"10.1016/j.xpro.2024.103580","DOIUrl":"10.1016/j.xpro.2024.103580","url":null,"abstract":"<p><p>The genome of the most recent common ancestor is generally not available but can greatly facilitate the inference of demographic history and the detection of local adaptations. Here, we present a protocol for applying local ancestry inference in present-day samples to reconstruct ancestral genomes. We describe steps for estimating haplotypes, inferring local ancestry, and assembling ancestral haplotypes. This protocol describes the analytic steps of reconstructing ancestral genomes using the example data of the Miao and She target populations. For complete details on the use and execution of this protocol, please refer to Gao et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103580"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11831122/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143013084","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 visualization of pRps6-positive cells in larval zebrafish brains using whole-mount immunofluorescence and light-sheet microscopy. 利用全载免疫荧光和薄层显微镜观察斑马鱼幼体大脑中prps6阳性细胞的方法。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-18 DOI: 10.1016/j.xpro.2024.103587
Olga Doszyn, Tomasz Dulski, Justyna Zmorzynska
{"title":"Protocol for the visualization of pRps6-positive cells in larval zebrafish brains using whole-mount immunofluorescence and light-sheet microscopy.","authors":"Olga Doszyn, Tomasz Dulski, Justyna Zmorzynska","doi":"10.1016/j.xpro.2024.103587","DOIUrl":"10.1016/j.xpro.2024.103587","url":null,"abstract":"<p><p>Due to their small size and transparency, larval zebrafish are a useful model for whole-brain imaging. Here, we present a protocol for the visualization of phosphorylated Rps6, a marker of mechanistic target of rapamycin complex 1 (mTORC1) activity, in the zebrafish brains at 5 days post fertilization (dpf), using whole-mount immunofluorescence and light-sheet microscopy. We describe steps for sample preparation, storage, staining, and imaging. This protocol can also be modified for staining with antibodies against other proteins. For complete details on the use and execution of this protocol, please refer to Doszyn et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103587"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11787575/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143013088","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 perform ex vivo electroretinography on adult zebrafish. 对成年斑马鱼进行离体视网膜电成像的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-16 DOI: 10.1016/j.xpro.2024.103565
Samantha L Simpson, Ross F Collery
{"title":"Protocol to perform ex vivo electroretinography on adult zebrafish.","authors":"Samantha L Simpson, Ross F Collery","doi":"10.1016/j.xpro.2024.103565","DOIUrl":"10.1016/j.xpro.2024.103565","url":null,"abstract":"<p><p>Ex vivo electroretinography (ERG) provides insight into the health and functionality of retinal cells, the integrity of phototransduction, and the visual cycle and allows for the direct application of pharmaceuticals to the retinal tissues. Here, we present a protocol for performing ex vivo ERG on adult zebrafish. We describe steps for zebrafish tissue dissection, mounting tissues, and assembling and connecting cassettes. We then detail procedures for running the Diagnosys software and processing and analyzing the resulting data.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103565"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11786734/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143013092","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 transcriptomic and epigenomic analyses of tip-like endothelial cells using scRNA-seq and ChIP-seq. 使用scRNA-seq和ChIP-seq对尖端样内皮细胞进行转录组学和表观基因组学分析的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-10 DOI: 10.1016/j.xpro.2024.103326
Shintaro Funasaki, Yuri Miyamura, Shunsuke Kamei, Akhinur Rahman, Masaya Yamazaki, Shingo Usuki, Keiichiro Yasunaga, Yorifumi Satou, Hiroto Ohguchi, Takashi Minami
{"title":"Protocol for transcriptomic and epigenomic analyses of tip-like endothelial cells using scRNA-seq and ChIP-seq.","authors":"Shintaro Funasaki, Yuri Miyamura, Shunsuke Kamei, Akhinur Rahman, Masaya Yamazaki, Shingo Usuki, Keiichiro Yasunaga, Yorifumi Satou, Hiroto Ohguchi, Takashi Minami","doi":"10.1016/j.xpro.2024.103326","DOIUrl":"10.1016/j.xpro.2024.103326","url":null,"abstract":"<p><p>Angiogenesis begins as endothelial cells migrate, forming a sprouting tip and subsequent growth-rich stalk cells. Here, we present a protocol for transcriptomic and epigenomic analyses of tip-like cells in cultured endothelial cells. We describe steps for stimulating human umbilical vein endothelial cells (HUVECs) with vascular endothelial cell growth factor (VEGF) to generate tip-like cells. We then detail procedures for library preparation for single-cell RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq), and data analysis. This scalable protocol is also applicable to diverse omics studies, including proteomics and metabolomics. For complete details on the use and execution of this protocol, please refer to Miyamura et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103326"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11772138/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972462","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 generate a 3D atherogenesis-on-chip model for studying endothelial-macrophage crosstalk in atherogenesis. 生成三维动脉粥样硬化芯片模型的方案，用于研究动脉粥样硬化中内皮-巨噬细胞串扰。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-11 DOI: 10.1016/j.xpro.2024.103559
Katie M L Hanford, Kim E Dzobo, Miranda Versloot, Jorge Peter, Jeffrey Kroon
{"title":"Protocol to generate a 3D atherogenesis-on-chip model for studying endothelial-macrophage crosstalk in atherogenesis.","authors":"Katie M L Hanford, Kim E Dzobo, Miranda Versloot, Jorge Peter, Jeffrey Kroon","doi":"10.1016/j.xpro.2024.103559","DOIUrl":"10.1016/j.xpro.2024.103559","url":null,"abstract":"<p><p>The endothelium is the gatekeeper of vessel health, and its dysfunction is pivotal in driving atherogenesis. Here, we present a protocol to replicate endothelial-macrophage crosstalk during atherogenesis, called the \"atherogenesis-on-chip\" model, based on the Emulate dual-channel perfusion system. We describe a model for studying endothelial-macrophage interactions during atherogenesis in human aortic endothelial cells and human macrophages using qPCR and secretome analysis, fluorescence microscopy, and flow cytometry. This protocol could be adapted toward more complex plaque microenvironment or other disease settings.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103559"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11773240/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142972463","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 isolating and purifying murine liver sinusoidal endothelial cells for in vitro culture and functional assays. 分离和纯化小鼠肝窦内皮细胞用于体外培养和功能测定的规程。
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-12 DOI: 10.1016/j.xpro.2024.103554
Sophia Papaioannou, Jia-Xiang See, Tinja Baljkas, Philipp Reiners-Koch, Manuel Winkler, Adelheid Cerwenka, Ana Stojanovic
{"title":"Protocol for isolating and purifying murine liver sinusoidal endothelial cells for in vitro culture and functional assays.","authors":"Sophia Papaioannou, Jia-Xiang See, Tinja Baljkas, Philipp Reiners-Koch, Manuel Winkler, Adelheid Cerwenka, Ana Stojanovic","doi":"10.1016/j.xpro.2024.103554","DOIUrl":"10.1016/j.xpro.2024.103554","url":null,"abstract":"<p><p>Liver sinusoidal endothelial cells (LSECs) line the liver sinusoids and play a crucial role in liver function. Isolating LSECs is beneficial for their functional evaluation in vitro. Here, we provide a protocol for obtaining purified LSECs from mice via gradient centrifugation and magnetic cell sorting (MACS), yielding cells suitable for culture and downstream analyses. We describe steps for culturing the purified LSECs and demonstrate their evaluation by flow cytometry. For complete details on the use and execution of this protocol, please refer to Papaioannou et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103554"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11772955/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142980211","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 boost the robustness and accuracy of spatial transcriptomics algorithms using ensemble techniques.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-28 DOI: 10.1016/j.xpro.2025.103608
Jiazhang Cai, Shushan Wu, Huimin Cheng, Wenxuan Zhong, Guo-Cheng Yuan, Ping Ma
{"title":"Protocol to boost the robustness and accuracy of spatial transcriptomics algorithms using ensemble techniques.","authors":"Jiazhang Cai, Shushan Wu, Huimin Cheng, Wenxuan Zhong, Guo-Cheng Yuan, Ping Ma","doi":"10.1016/j.xpro.2025.103608","DOIUrl":"10.1016/j.xpro.2025.103608","url":null,"abstract":"<p><p>Spatial transcriptomics enhances our understanding of cellular organization by mapping gene expression data to precise tissue locations. Here, we present a protocol for using weighted ensemble method for spatial transcriptomics (WEST), which uses ensemble techniques to boost the robustness and accuracy of existing algorithms. We describe steps for preprocessing data, obtaining embeddings from individual algorithms, and ensemble integrating all embeddings as a similarity matrix. We then detail procedures for using the similarity matrix to identify spatial domains and obtain new embeddings. For complete details on the use and execution of this protocol, please refer to Cai et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103608"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11803146/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143068428","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 profiling RNA m5C methylation at base resolution using m5C-TAC-seq.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-01-31 DOI: 10.1016/j.xpro.2025.103599
Xiaoting Zhang, Liang Lu, Chengqi Yi, Xiaoyu Li
{"title":"Protocol for profiling RNA m<sup>5</sup>C methylation at base resolution using m<sup>5</sup>C-TAC-seq.","authors":"Xiaoting Zhang, Liang Lu, Chengqi Yi, Xiaoyu Li","doi":"10.1016/j.xpro.2025.103599","DOIUrl":"10.1016/j.xpro.2025.103599","url":null,"abstract":"<p><p>RNA 5-methylcytosine (m<sup>5</sup>C) is a widespread modification and plays a crucial role in gene expression regulation. Here, we present a protocol for transcriptome-wide m<sup>5</sup>C methylome profiling at base resolution using bisulfite-free m<sup>5</sup>C detection strategy enabled by ten-eleven translocation (TET)-assisted chemical labeling sequencing (m<sup>5</sup>C-TAC-seq). We detail steps for TET-assisted chemical labeling, library construction, and data analysis. m<sup>5</sup>C-TAC-seq enables accurate and robust m<sup>5</sup>C detection in various RNA species. For complete details on the use and execution of this protocol, please refer to Lu et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103599"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11835647/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143081356","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 direct cDNA cap analysis of gene expression for paired-end patterned flow cell sequencing.
IF 1.3
STAR Protocols Pub Date : 2025-03-21 Epub Date: 2025-02-06 DOI: 10.1016/j.xpro.2024.103594
Diane Delobel, Hiromi Nishiyori-Sueki, Ilaria Nisoli, Hideya Kawaji, Pauline Robbe, Piero Carninci, Hazuki Takahashi
{"title":"Protocol for direct cDNA cap analysis of gene expression for paired-end patterned flow cell sequencing.","authors":"Diane Delobel, Hiromi Nishiyori-Sueki, Ilaria Nisoli, Hideya Kawaji, Pauline Robbe, Piero Carninci, Hazuki Takahashi","doi":"10.1016/j.xpro.2024.103594","DOIUrl":"10.1016/j.xpro.2024.103594","url":null,"abstract":"<p><p>Cap analysis of gene expression (CAGE) is a technique that facilitates the assessment of the 5'-end of RNA transcript starting site (TSS) of both coding and non-coding genes. Here, we present a protocol for using CAGE on Illumina patterned flow cell technology with dual indexes on mouse and human samples. We describe steps for sequencing, automated data processing, and complete analytical framework ensuring CAGE operability for the determination of TSS and enhancers on the ever-evolving Illumina sequencing platforms.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 1","pages":"103594"},"PeriodicalIF":1.3,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11851279/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143374761","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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