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Protocol for achieving enhanced snRNA-seq data quality using Quality Clustering.
IF 1.3
STAR Protocols Pub Date : 2025-03-28 DOI: 10.1016/j.xpro.2025.103717
Paavo J Tavi, Johannes Ojanen, Pia Laitinen, Suvi Linna-Kuosmanen
{"title":"Protocol for achieving enhanced snRNA-seq data quality using Quality Clustering.","authors":"Paavo J Tavi, Johannes Ojanen, Pia Laitinen, Suvi Linna-Kuosmanen","doi":"10.1016/j.xpro.2025.103717","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103717","url":null,"abstract":"<p><p>Single-nucleus RNA sequencing (snRNA-seq) data analysis presents a challenge in samples that have high levels of ambient RNA contamination. Quality Clustering (QClus) removes empty and highly contaminated droplets by utilizing multiple contamination metrics. Here, we present the steps for snRNA-seq data preprocessing using the QClus algorithm. First, we describe how to set up a computational environment. Next, we demonstrate how to use QClus to remove highly contaminated droplets, and finally, we show how to visualize and evaluate the results. For complete details on the use and execution of this protocol, please refer to Schmauch et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103717"},"PeriodicalIF":1.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143754785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for high-throughput screening of SIRT7 inhibitors using fluorescent peptide technology. 利用荧光肽技术高通量筛选 SIRT7 抑制剂的方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-28 DOI: 10.1016/j.xpro.2025.103720
Tian-Shu Kang, Xiaopeng Lu, Jinke Gu, Yuan Tian, Wei-Guo Zhu
{"title":"Protocol for high-throughput screening of SIRT7 inhibitors using fluorescent peptide technology.","authors":"Tian-Shu Kang, Xiaopeng Lu, Jinke Gu, Yuan Tian, Wei-Guo Zhu","doi":"10.1016/j.xpro.2025.103720","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103720","url":null,"abstract":"<p><p>Fluorescent peptides combine the polypeptides with fluorescent groups organically to facilitate the measurement of enzyme activity. Here, we present a protocol to evaluate Sirtuin 7 (SIRT7) enzymatic activity by analyzing changes in luminescent signals from its substrate polypeptides. We outline the steps for SIRT7 protein purification, enzymatic reaction setup, fluorescence spectrum detection, and data analysis. Additionally, we describe two applications targeting SIRT7: high-throughput screening of SIRT7 inhibitors using microplate and the validation of potential SIRT7 inhibitors. For complete details on the use and execution of this protocol, please refer to Kang et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103720"},"PeriodicalIF":1.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143744092","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for producing brain-derived neurotrophic factor and neurotrophin-4 in their pro and active form in Escherichia coli.
IF 1.3
STAR Protocols Pub Date : 2025-03-28 DOI: 10.1016/j.xpro.2025.103715
Christoph Holzner, Katharina Böttinger, Christian G Huber, Elfriede Dall, Hans Brandstetter
{"title":"Protocol for producing brain-derived neurotrophic factor and neurotrophin-4 in their pro and active form in Escherichia coli.","authors":"Christoph Holzner, Katharina Böttinger, Christian G Huber, Elfriede Dall, Hans Brandstetter","doi":"10.1016/j.xpro.2025.103715","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103715","url":null,"abstract":"<p><p>Brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT4) are neurotrophic growth factors, signaling primarily via the tropomyosin receptor kinase B (TrkB). Here, we present a protocol for producing BDNF and NT4 in their pro and active form in Escherichia coli. We describe steps for E. coli expression, oxidative in vitro folding from inclusion bodies, and purification. We then detail procedures for proteolytic activation and characterization of the purified neurotrophins. For complete details on the use and execution of this protocol, please refer to Holzner et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103715"},"PeriodicalIF":1.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143754850","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for constructing in vivo and in vitro models integrating clinical and proteomic subtypes of chordoma. 整合脊索瘤临床亚型和蛋白质组亚型的体内和体外模型构建方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-28 DOI: 10.1016/j.xpro.2025.103719
Jianxuan Gao, Jinbo Hu, Tong Meng
{"title":"Protocol for constructing in vivo and in vitro models integrating clinical and proteomic subtypes of chordoma.","authors":"Jianxuan Gao, Jinbo Hu, Tong Meng","doi":"10.1016/j.xpro.2025.103719","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103719","url":null,"abstract":"<p><p>Here, we present a protocol for constructing both in vivo and in vitro models of chordoma, integrating clinical and proteomic subtyping to enable a comprehensive analysis. We detail the steps for tissue sample preparation and tandem mass tag proteomic analysis and apply non-negative matrix factorization for molecular classification. Furthermore, we classify chordoma cell lines and establish cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models in mice for investigating drug response. For complete details on the use and execution of this protocol, please refer to Yin et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103719"},"PeriodicalIF":1.3,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143744091","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for spatial metabolomics and isotope tracing in the mouse brain. 小鼠大脑空间代谢组学和同位素追踪方案。
IF 1.3
STAR Protocols Pub Date : 2025-03-27 DOI: 10.1016/j.xpro.2025.103716
Watit Sontising, Francine Yanchik-Slade, Carlos Rodriguez-Navas, Md Amir Hossen, Manuel Gonzalez-Rodriguez, Jake Vaynshteyn, Shinichi Yamaguchi, Mohamed Nazim Boutaghou, Isaac Marin-Valencia
{"title":"Protocol for spatial metabolomics and isotope tracing in the mouse brain.","authors":"Watit Sontising, Francine Yanchik-Slade, Carlos Rodriguez-Navas, Md Amir Hossen, Manuel Gonzalez-Rodriguez, Jake Vaynshteyn, Shinichi Yamaguchi, Mohamed Nazim Boutaghou, Isaac Marin-Valencia","doi":"10.1016/j.xpro.2025.103716","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103716","url":null,"abstract":"<p><p>Mass spectrometry imaging enables high-resolution spatial chemical mapping, yet its application for dynamic analysis with tracers poses challenges. Here, we present a protocol for spatial metabolomics and isotope tracing in the mouse brain. We describe steps for tracer administration, tissue collection, and cryosectioning. We then detail procedures for matrix application, ion identification, and data analysis. This protocol delivers high-quality spatial metabolomics data and is well suited for region-specific tracing analysis in the brain.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103716"},"PeriodicalIF":1.3,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143735892","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for biomimetic tumoroid models by plastic compression using centrifugation.
IF 1.3
STAR Protocols Pub Date : 2025-03-26 DOI: 10.1016/j.xpro.2025.103718
Sam Devereaux, Ashley Lam, Anuja Upadhyay, Megan Fallows, Lianqi Qiu, Xueni Pan, Umber Cheema
{"title":"Protocol for biomimetic tumoroid models by plastic compression using centrifugation.","authors":"Sam Devereaux, Ashley Lam, Anuja Upadhyay, Megan Fallows, Lianqi Qiu, Xueni Pan, Umber Cheema","doi":"10.1016/j.xpro.2025.103718","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103718","url":null,"abstract":"<p><p>Here, we present a protocol for engineering biomimetic tumoroid models by plastic compression using centrifugation. We describe steps for generating multi-compartment tumor-stroma models by mixing cells into a collagen hydrogel crosslinked at 37°C and centrifuging the hydrogel. We then detail procedures for generating compartmentalized models and encapsulating the final layered hydrogel containing a 96-well tumor mass in a 24-well stroma. This protocol increases collagen density and improves mechanical properties of collagen hydrogels.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103718"},"PeriodicalIF":1.3,"publicationDate":"2025-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143731929","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for investigating the impact of transcription regulator deficiency on tumor-specific CD8+ T cell responses via adoptive cell transfer.
IF 1.3
STAR Protocols Pub Date : 2025-03-25 DOI: 10.1016/j.xpro.2025.103703
Luming Xu, Yuzhu Wang, Mengqu Ran, Wen Liu, Lilin Ye, Qizhao Huang
{"title":"Protocol for investigating the impact of transcription regulator deficiency on tumor-specific CD8<sup>+</sup> T cell responses via adoptive cell transfer.","authors":"Luming Xu, Yuzhu Wang, Mengqu Ran, Wen Liu, Lilin Ye, Qizhao Huang","doi":"10.1016/j.xpro.2025.103703","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103703","url":null,"abstract":"<p><p>Transcription factors play a crucial role in the differentiation of tumor-specific CD8<sup>+</sup> T cells and significantly influence their capacity to repress tumors. Here, we present a protocol for creating a transcription regulator inhibitor of DNA 3 (ID3) conditional knockout mouse in tumor-specific CD8<sup>+</sup> T cells, induced by tamoxifen. We describe steps for examining the impact of ID3 deficiency on the differentiation of tumor-specific memory CD8<sup>+</sup> T cells (Ttsm) and progenitors of exhausted CD8<sup>+</sup> T cells (Tpex) in tumor-draining lymph nodes through a co-adoptive transfer assay. For complete details on the use and execution of this protocol, please refer to Ran et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103703"},"PeriodicalIF":1.3,"publicationDate":"2025-03-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143731930","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for the development, assembly, and testing of a synthetic skin microbial community.
IF 1.3
STAR Protocols Pub Date : 2025-03-24 DOI: 10.1016/j.xpro.2025.103714
Deepan Thiruppathy, Asama Lekbua, Joanna Coker, Yuhan Weng, Fatemeh Askarian, Armin Kousha, Clarisse Marotz, Amber Hauw, Megan Tjuanta, Victor Nizet, Karsten Zengler
{"title":"Protocol for the development, assembly, and testing of a synthetic skin microbial community.","authors":"Deepan Thiruppathy, Asama Lekbua, Joanna Coker, Yuhan Weng, Fatemeh Askarian, Armin Kousha, Clarisse Marotz, Amber Hauw, Megan Tjuanta, Victor Nizet, Karsten Zengler","doi":"10.1016/j.xpro.2025.103714","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103714","url":null,"abstract":"<p><p>A reproducible study system is essential for understanding the role of microbes in human skin health and disease. We present a protocol for constructing a synthetic microbial community (SkinCom) of nine strains dominant to native human skin microbiome. We describe steps for computing growth metrics, constructing communities, and extracting DNA and library preparation for shotgun sequencing. We detail steps for data preprocessing and analysis of community samples. We illustrate SkinCom's application with an epicutaneous murine model and downstream multiomic analysis. For complete details on the use and execution of this protocol, please refer to Lekbua et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103714"},"PeriodicalIF":1.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143731931","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Protocol for evaluating neuronal activity and neurotransmitter release following amyloid-beta oligomer injections into the rat hippocampus.
IF 1.3
STAR Protocols Pub Date : 2025-03-24 DOI: 10.1016/j.xpro.2025.103712
Vincent Hervé, Laurie Bonenfant, Mathilde Amyot, Rime Balafrej, Obai Bin Ka'B Ali, Habib Benali, Jonathan Brouillette
{"title":"Protocol for evaluating neuronal activity and neurotransmitter release following amyloid-beta oligomer injections into the rat hippocampus.","authors":"Vincent Hervé, Laurie Bonenfant, Mathilde Amyot, Rime Balafrej, Obai Bin Ka'B Ali, Habib Benali, Jonathan Brouillette","doi":"10.1016/j.xpro.2025.103712","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103712","url":null,"abstract":"<p><p>In Alzheimer's disease, there is an imbalance in neurotransmitter release and altered neuronal activation. Here, we present a protocol approach to analyze neuronal activity by combining local field potential (LFP) recording with microdialysis within the same animal. We describe steps for measuring glutamate and GABA levels following hippocampal amyloid-beta oligomer (Aβo) injections in rats. We then detail procedures for assembling the electrode and cannula, surgical implantation and simultaneous in vivo LFP recording, interstitial fluid collection, and Aβo injections.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103712"},"PeriodicalIF":1.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143711434","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
An optimized protocol for generating fibrotic murine precision-cut lung slices.
IF 1.3
STAR Protocols Pub Date : 2025-03-24 DOI: 10.1016/j.xpro.2025.103711
Stefanos Smyrniotis, Paraskevi Kanellopoulou, Christiana Magkrioti, Vassilis Aidinis
{"title":"An optimized protocol for generating fibrotic murine precision-cut lung slices.","authors":"Stefanos Smyrniotis, Paraskevi Kanellopoulou, Christiana Magkrioti, Vassilis Aidinis","doi":"10.1016/j.xpro.2025.103711","DOIUrl":"https://doi.org/10.1016/j.xpro.2025.103711","url":null,"abstract":"<p><p>Precision-cut lung slices (PCLSs) offer significant advantages over both in vivo animal models and in vitro cell models, making them invaluable tools in respiratory research. Here, we present a protocol for generating fibrotic mouse PCLSs (mPCLSs), which offer the same benefits as human PCLSs but bypass issues of human tissue availability and related ethical concerns while also enhancing experimental homogeneity and reproducibility. This protocol details the steps for bleomycin (BLM)-induced pulmonary fibrosis, PCLS generation, visualization, and downstream analysis. For complete details on the use and execution of this protocol, please refer to Barbayianni et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"6 2","pages":"103711"},"PeriodicalIF":1.3,"publicationDate":"2025-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143731927","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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