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Protocol for genome-wide differential methylation using long-read DNA sequencing in the cortex of a mouse model of Alzheimer's disease. 在阿尔茨海默病小鼠模型的皮层中使用长读DNA测序进行全基因组差异甲基化的方案。
IF 1.4
STAR Protocols Pub Date : 2026-10-08 DOI: 10.1016/j.xpro.2026.104827
Ümran Yaman, Emil Gustavsson, Jasmaine Lee, Hermione Ugolini, Hannah Macpherson, Paige Mumford, Frances Wiseman, John Hardy, Dervis A Salih
{"title":"Protocol for genome-wide differential methylation using long-read DNA sequencing in the cortex of a mouse model of Alzheimer's disease.","authors":"Ümran Yaman, Emil Gustavsson, Jasmaine Lee, Hermione Ugolini, Hannah Macpherson, Paige Mumford, Frances Wiseman, John Hardy, Dervis A Salih","doi":"10.1016/j.xpro.2026.104827","DOIUrl":"10.1016/j.xpro.2026.104827","url":null,"abstract":"<p><p>Third-generation sequencing enables direct detection of genome-wide 5-methylcytosine without bisulfite conversion. Here, we present a protocol for generating long-read DNA sequencing from mouse cortex and performing genome-wide differential methylation analysis. We describe steps for high-molecular-weight DNA extraction, data generation, and downstream analysis. We demonstrate the workflow in the App<sup>NL-G-F</sup> mouse model of amyloid pathology and provide a reproducible pipeline that links modified-base BAM (modBAM) files to annotated differentially methylated regions and functional enrichment results.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104827"},"PeriodicalIF":1.4,"publicationDate":"2026-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13675703/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149149222","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 scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration. 用于wnt信号激活和组织再生的可口服r -spondin1负载小细胞外囊泡的规模化生产方案。
IF 1.4
STAR Protocols Pub Date : 2026-10-08 DOI: 10.1016/j.xpro.2026.104891
Lingyan Yang, Ting Jiang, Shixiang Wang, Yue Yang, Zhiping Qiao, Yun-Shen Chan
{"title":"Protocol for scalable production of orally deliverable R-spondin1-loaded small extracellular vesicles for WNT-signaling activation and tissue regeneration.","authors":"Lingyan Yang, Ting Jiang, Shixiang Wang, Yue Yang, Zhiping Qiao, Yun-Shen Chan","doi":"10.1016/j.xpro.2026.104891","DOIUrl":"10.1016/j.xpro.2026.104891","url":null,"abstract":"<p><p>R-spondin1 plays a pivotal role in potentiating canonical WNT/β-catenin signaling, a pathway critical for maintaining stem cell homeostasis and driving tissue regeneration. Here, we present a scalable, serum-free protocol for producing R-spondin1-loaded small extracellular vesicles (evRSPO1) from engineered HEK293 cells, with multimodal characterization of vesicle quality and bioactivity. We also describe steps for in vitro functional validation of the vesicles using mouse intestinal organoids and for in vivo therapeutic applications in mouse intestinal regeneration via oral delivery. For complete details on the use and execution of this protocol, please refer to Yang et al.<sup>1</sup><sup>,</sup><sup>2</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104891"},"PeriodicalIF":1.4,"publicationDate":"2026-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13676708/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149138520","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 establishing a murine Toscana virus infection model and downstream cellular analysis of skin tissue and cells. 建立小鼠托斯卡纳病毒感染模型及皮肤组织和细胞下游细胞分析方案。
IF 1.4
STAR Protocols Pub Date : 2026-10-08 DOI: 10.1016/j.xpro.2026.104885
Yonca Keskek Turk, Ailish McCafferty-Brown, Clive S McKimmie
{"title":"Protocol for establishing a murine Toscana virus infection model and downstream cellular analysis of skin tissue and cells.","authors":"Yonca Keskek Turk, Ailish McCafferty-Brown, Clive S McKimmie","doi":"10.1016/j.xpro.2026.104885","DOIUrl":"10.1016/j.xpro.2026.104885","url":null,"abstract":"<p><p>Here, we present an in vivo approach to study virus-vector-host interactions during Toscana virus (TOSV) infection in mouse skin. We describe preparation of viral stocks and subcutaneous TOSV inoculation into dorsal upper foot skin, either alone, with sand-fly salivary gland extract (SGE), or following sand-fly biting. We then detail tissue collection and generation of single-cell suspensions. Finally, we describe flow cytometric detection of infected cells, magnetic isolation of dermal fibroblasts, and RNA extraction for transcriptomic analysis. For complete details on the use and execution of this protocol, please refer to Keskek Turk et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104885"},"PeriodicalIF":1.4,"publicationDate":"2026-10-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13675714/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149138470","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 quantifying chromatin DNA replication by multiplex labeling and single-molecule localization microscopy. 多重标记和单分子定位显微镜定量染色质DNA复制的方法。
IF 1.4
STAR Protocols Pub Date : 2026-10-07 DOI: 10.1016/j.xpro.2026.104882
Zhiguang Xiao, Xiaodong Guan, Yujie Sun, Mengling Zhang, Qian Peter Su
{"title":"Protocol for quantifying chromatin DNA replication by multiplex labeling and single-molecule localization microscopy.","authors":"Zhiguang Xiao, Xiaodong Guan, Yujie Sun, Mengling Zhang, Qian Peter Su","doi":"10.1016/j.xpro.2026.104882","DOIUrl":"10.1016/j.xpro.2026.104882","url":null,"abstract":"<p><p>We present a protocol for imaging and quantifying chromatin DNA replication-associated signals in synchronized mammalian cells. We describe steps for cell-cycle enrichment, nucleoside analog-based metabolic labeling, and fluorescence detection. We then detail the procedure for dual-color STORM imaging and SR-Tesseler-based cluster analysis. It can be adapted to distinct questions in DNA replication biology by modifying synchronization schemes, pulse durations, labeling strategies, fluorescence detection channels, or analysis parameters. For complete details on the use and execution of this protocol, please refer to Zhang et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104882"},"PeriodicalIF":1.4,"publicationDate":"2026-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13676695/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149128574","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 isolation and cryopreservation of human peripheral blood mononuclear cells for high-resolution respirometry. 用于高分辨率呼吸测量的人外周血单个核细胞的分离和低温保存方案。
IF 1.4
STAR Protocols Pub Date : 2026-10-07 DOI: 10.1016/j.xpro.2026.104881
Alejandra Romero, Alba Timón-Gómez, Jaime R Willis, Anja Perfler, Theresa Lindlbauer, Michael Knoflach, Erich Gnaiger
{"title":"Protocol for isolation and cryopreservation of human peripheral blood mononuclear cells for high-resolution respirometry.","authors":"Alejandra Romero, Alba Timón-Gómez, Jaime R Willis, Anja Perfler, Theresa Lindlbauer, Michael Knoflach, Erich Gnaiger","doi":"10.1016/j.xpro.2026.104881","DOIUrl":"10.1016/j.xpro.2026.104881","url":null,"abstract":"<p><p>High-resolution respirometry (HRR) of peripheral blood mononuclear cells (PBMCs) relies on controlled preanalytical procedures. Here, we present an optimized workflow for PBMC isolation from human whole blood, followed by short-term cryopreservation at -80°C. The protocol includes rapid thawing to preserve cell viability, washing steps to minimize platelet contamination, and quality-control assessment of PBMC number, viability, and composition before HRR measurements. Critical handling steps are optimized and validated by HRR against freshly prepared cells.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104881"},"PeriodicalIF":1.4,"publicationDate":"2026-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13676717/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149128602","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 high-throughput identification of genes involved in pyroptotic cell death by live microscopy. 活体显微镜下高通量鉴定热噬细胞死亡相关基因的规程。
IF 1.4
STAR Protocols Pub Date : 2026-10-07 DOI: 10.1016/j.xpro.2026.104884
Aleksandra Bajger, Lukasz S Borowski, Roman J Szczesny, Michal P Wandel
{"title":"Protocol for high-throughput identification of genes involved in pyroptotic cell death by live microscopy.","authors":"Aleksandra Bajger, Lukasz S Borowski, Roman J Szczesny, Michal P Wandel","doi":"10.1016/j.xpro.2026.104884","DOIUrl":"10.1016/j.xpro.2026.104884","url":null,"abstract":"<p><p>Intracellular lipopolysaccharide activates caspase-4/5 triggering pyroptotic cell death. We present a protocol for a high-throughput loss-of-function screening approach to identify interferon-gamma-stimulated genes regulating this pathway. We detail procedures for cell reverse-transfection with a custom-made small interfering RNA (siRNA) library in 384-well plates and priming with interferon-gamma. We then describe steps for transfection with lipopolysaccharide to induce pyroptosis and live-cell fluorescence microscopy using the IncuCyte S3 system to quantify cell death in real time without fixation. This approach enables reliable, high-throughput screening of cell death.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104884"},"PeriodicalIF":1.4,"publicationDate":"2026-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13676704/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149138542","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 mapping sister chromatid exchanges and structural variations using sci-L3-strand-seq. 使用sci- l3 -链序列绘制姐妹染色单体交换和结构变化的方案。
IF 1.4
STAR Protocols Pub Date : 2026-10-07 DOI: 10.1016/j.xpro.2026.104892
Shiyang He, Peter Chovanec, Yi Yin
{"title":"Protocol for mapping sister chromatid exchanges and structural variations using sci-L3-strand-seq.","authors":"Shiyang He, Peter Chovanec, Yi Yin","doi":"10.1016/j.xpro.2026.104892","DOIUrl":"10.1016/j.xpro.2026.104892","url":null,"abstract":"<p><p>Understanding spontaneous genome rearrangement requires mapping both error-free and mutational repair outcomes, particularly error-free sister chromatid exchanges (SCEs). Here, we present a sci-L3-Strand-seq protocol capturing SCEs across thousands of single cells from 12-16 independent samples in one experiment. We detail steps for cell labeling, sample banking, nucleosome depletion, in situ combinatorial barcoding, enrichment of BrdU-labeled cells, and nascent strand ablation. We then detail linear amplification, reverse transcription, and second-strand synthesis. Libraries are typically sequencing-ready within three days from frozen cells. For complete details on the use and execution of this protocol, please refer to Chovanec et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104892"},"PeriodicalIF":1.4,"publicationDate":"2026-10-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13676703/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149138475","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 Cas9 nickase-mediated induction and two-dimensional gel detection of i-loops at human telomeric and alpha satellite repeats. Cas9镍酶介导的人类端粒和α卫星重复序列i环诱导和二维凝胶检测方案
IF 1.4
STAR Protocols Pub Date : 2026-10-06 DOI: 10.1016/j.xpro.2026.104888
Elia Zanella, Flavia Bruno, Ylli Doksani
{"title":"Protocol for Cas9 nickase-mediated induction and two-dimensional gel detection of i-loops at human telomeric and alpha satellite repeats.","authors":"Elia Zanella, Flavia Bruno, Ylli Doksani","doi":"10.1016/j.xpro.2026.104888","DOIUrl":"10.1016/j.xpro.2026.104888","url":null,"abstract":"<p><p>Single-stranded DNA breaks (SSBs) at repetitive elements promote internal loops (i-loops), precursors of extrachromosomal circular DNA (eccDNA). Here, we present a protocol for inducing targeted breaks at human telomeric or alpha-satellite repeats using doxycycline-inducible Cas9 nickase variants and repeat-specific sgRNAs. We describe steps for verifying strand-specific nicking by alkaline-gel Southern blotting and detecting i-loop accumulation by two-dimensional agarose gel electrophoresis (2D gel). In this protocol, DNase I treatment of isolated nuclei is employed as a Cas9-independent control. For complete details on the use and execution of this protocol, please refer to Zanella et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104888"},"PeriodicalIF":1.4,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13675476/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149115791","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 analyzing tumor-infiltrating bystander CD8+ T cells in mice using adoptive transfer of P14 TCR-transgenic CD8+ T cells. 使用P14 tcr转基因CD8+ T细胞过继转移分析小鼠肿瘤浸润的旁观者CD8+ T细胞的方案。
IF 1.4
STAR Protocols Pub Date : 2026-10-06 DOI: 10.1016/j.xpro.2026.104886
Chunyang Xie, Wenwen Xi, Hairu Wang, Lilin Ye, Xiangyu Chen, Yao Lin
{"title":"Protocol for analyzing tumor-infiltrating bystander CD8<sup>+</sup> T cells in mice using adoptive transfer of P14 TCR-transgenic CD8<sup>+</sup> T cells.","authors":"Chunyang Xie, Wenwen Xi, Hairu Wang, Lilin Ye, Xiangyu Chen, Yao Lin","doi":"10.1016/j.xpro.2026.104886","DOIUrl":"10.1016/j.xpro.2026.104886","url":null,"abstract":"<p><p>While tumor-specific CD8<sup>+</sup> T cells become functionally exhausted within the tumor microenvironment (TME), virus-specific bystander memory CD8<sup>+</sup> T (T<sub>BYS</sub>) cells are abundant and represent valuable immunotherapy targets. Here, we present a protocol to analyze tumor-infiltrating virus-specific T<sub>BYS</sub> cells in murine tumor models using P14 CD8<sup>+</sup> T cells. We describe steps for monitoring long-term in vivo behavior of P14 T<sub>BYS</sub> cells and characterizing their differentiation trajectories and functional heterogeneity. For complete details on the use and execution of this protocol, please refer to Lin et al.<sup>1</sup>.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104886"},"PeriodicalIF":1.4,"publicationDate":"2026-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13672856/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149115789","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 chronic electroencephalography, fiber photometry, and silicon probe recordings in the Syrian hamster. 叙利亚仓鼠慢性脑电图、纤维光度测定和硅探针记录的方案。
IF 1.4
STAR Protocols Pub Date : 2026-10-05 DOI: 10.1016/j.xpro.2026.104863
Fuki Yamashita, Ren Y Sato, Yasutaka Mukai, Hiroaki Nishimura, Sarah Dagher, Shimpei Ishiyama, Miyu Kimata, Riho Kobayashi, Hiroaki Norimoto
{"title":"Protocol for chronic electroencephalography, fiber photometry, and silicon probe recordings in the Syrian hamster.","authors":"Fuki Yamashita, Ren Y Sato, Yasutaka Mukai, Hiroaki Nishimura, Sarah Dagher, Shimpei Ishiyama, Miyu Kimata, Riho Kobayashi, Hiroaki Norimoto","doi":"10.1016/j.xpro.2026.104863","DOIUrl":"10.1016/j.xpro.2026.104863","url":null,"abstract":"<p><p>The Syrian hamster (Mesocricetus auratus) exhibits a unique metabolic physiology, including the ability to enter hypometabolic states. While this species represents a powerful model for studying the neuronal basis of metabolic control and brain-state transitions, its use is limited due to the lack of detailed protocols for chronic neuronal measurements. Here, we present a step-by-step protocol for chronic surgical implantation and multimodal neuronal recordings in Syrian hamsters. We describe steps for electroencephalography (EEG), fiber photometry, and multi-channel silicon probe recordings.</p>","PeriodicalId":34214,"journal":{"name":"STAR Protocols","volume":"7 4","pages":"104863"},"PeriodicalIF":1.4,"publicationDate":"2026-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13672980/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149115769","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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