Darian T Carroll, Sarah R Lindsley, Melissa Kirigiti, Alex Buko, Angela Jones, Paul Kievit, Maureen Gannon
{"title":"制备冷冻的非人灵长类胎儿胰岛,用于单核 RNA 测序、ATAC 测序和大容量代谢组学分析。","authors":"Darian T Carroll, Sarah R Lindsley, Melissa Kirigiti, Alex Buko, Angela Jones, Paul Kievit, Maureen Gannon","doi":"10.3791/66849","DOIUrl":null,"url":null,"abstract":"<p><p>One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, such as combined single-cell RNA sequencing and metabolomics. The method in the current study utilizes flash-frozen pancreatic islets from fetal non-human primates collected over a span of two years for input into single-nucleus RNA sequencing and ATAC sequencing assays. The cytosolic fraction generated during nuclear extraction was retained for downstream capillary electrophoresis-mass spectrometry and subsequent metabolite quantification. This method allows for bulk analysis of metabolites that contribute to the changing transcriptomic and epigenomic landscapes within experimental conditions. It is applicable to many tissue types and maximizes the amount of information that can be extracted from samples that are not readily available. As the contribution of metabolism to the establishment of cellular identity via epigenetic modifications becomes more appreciated, techniques that allow for identifying the contribution of metabolites in specific cell types are timely and necessary.</p>","PeriodicalId":48787,"journal":{"name":"Jove-Journal of Visualized Experiments","volume":" 213","pages":""},"PeriodicalIF":1.2000,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics.\",\"authors\":\"Darian T Carroll, Sarah R Lindsley, Melissa Kirigiti, Alex Buko, Angela Jones, Paul Kievit, Maureen Gannon\",\"doi\":\"10.3791/66849\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, such as combined single-cell RNA sequencing and metabolomics. The method in the current study utilizes flash-frozen pancreatic islets from fetal non-human primates collected over a span of two years for input into single-nucleus RNA sequencing and ATAC sequencing assays. The cytosolic fraction generated during nuclear extraction was retained for downstream capillary electrophoresis-mass spectrometry and subsequent metabolite quantification. This method allows for bulk analysis of metabolites that contribute to the changing transcriptomic and epigenomic landscapes within experimental conditions. It is applicable to many tissue types and maximizes the amount of information that can be extracted from samples that are not readily available. As the contribution of metabolism to the establishment of cellular identity via epigenetic modifications becomes more appreciated, techniques that allow for identifying the contribution of metabolites in specific cell types are timely and necessary.</p>\",\"PeriodicalId\":48787,\"journal\":{\"name\":\"Jove-Journal of Visualized Experiments\",\"volume\":\" 213\",\"pages\":\"\"},\"PeriodicalIF\":1.2000,\"publicationDate\":\"2024-11-08\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Jove-Journal of Visualized Experiments\",\"FirstCategoryId\":\"103\",\"ListUrlMain\":\"https://doi.org/10.3791/66849\",\"RegionNum\":4,\"RegionCategory\":\"综合性期刊\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"MULTIDISCIPLINARY SCIENCES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Jove-Journal of Visualized Experiments","FirstCategoryId":"103","ListUrlMain":"https://doi.org/10.3791/66849","RegionNum":4,"RegionCategory":"综合性期刊","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"MULTIDISCIPLINARY SCIENCES","Score":null,"Total":0}
Preparation of Frozen Non-Human Primate Fetal Islets for Combined Single Nuclei RNA-Sequencing and ATAC-Sequencing, and Bulk Metabolomics.
One challenge in studies using tissue collected from multiple cohorts is avoiding batch effects when preparing for large-scale multi-omic experiments, such as combined single-cell RNA sequencing and metabolomics. The method in the current study utilizes flash-frozen pancreatic islets from fetal non-human primates collected over a span of two years for input into single-nucleus RNA sequencing and ATAC sequencing assays. The cytosolic fraction generated during nuclear extraction was retained for downstream capillary electrophoresis-mass spectrometry and subsequent metabolite quantification. This method allows for bulk analysis of metabolites that contribute to the changing transcriptomic and epigenomic landscapes within experimental conditions. It is applicable to many tissue types and maximizes the amount of information that can be extracted from samples that are not readily available. As the contribution of metabolism to the establishment of cellular identity via epigenetic modifications becomes more appreciated, techniques that allow for identifying the contribution of metabolites in specific cell types are timely and necessary.
期刊介绍:
JoVE, the Journal of Visualized Experiments, is the world''s first peer reviewed scientific video journal. Established in 2006, JoVE is devoted to publishing scientific research in a visual format to help researchers overcome two of the biggest challenges facing the scientific research community today; poor reproducibility and the time and labor intensive nature of learning new experimental techniques.