DNA Isolation Methodology for Microbiome Genomics
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PROVIDER: PRJEB14814 | ENA |
REPOSITORIES: ENA
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Project description:Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition
| PRJEB12431 | ENA
Project description:Background: Microglial isolation and culturing methods continue to be explored to maximize cellular yield, purity, responsiveness to stimulation and similarity to in vivo microglia. This study aims to evaluate different microglia isolation methods — 3 variants of microglia isolation from neonatal mice and 2 variants of microglia isolation from adult mice — on transcriptional profile and response to HMGB1. Methods: Microglia from neonatal mice, age 0-3 days (P0-P3) were isolated from mixed glial cultures (MGC). We included three variations of this protocol that differed by use of GM-CSF in culture (No GM-CSF or 500 pg/mL GM-CSF), and days of culture in MGC before microglial separation (10 or 21). Protocols for studying microglia from adult mice age 6-8 weeks included isolation by adherence properties followed by 7 days of culture with 100 ng/mL GM-CSF and 100 ng/mL M-CSF (Vijaya et al., 2023), or acute isolation using CD11b beads (Bordt et al., 2020). Purity, yield, and RNA quality of the isolated microglia were assessed by flow cytometry, hemocytometer counting, Nanodrop, and Bioanalyzer, respectively. Microglial responsiveness to an inflammatory stimulus, HMGB1, was evaluated by measuring TNFα, IL1β, and IFNβ secretion by ELISA and assessing gene expression patterns using bulk RNA sequencing. Results: All five methods demonstrated greater than 90% purity. Microglia from all cultures increased transcription of or secreted TNFα, IL1β, and IFNβ in response to HMGB1. RNA sequencing showed a larger number of differentially expressed genes in response to HMGB1 treatment in microglia cultured from pups than from adult mice, with sparse changes among the 3 MGC culturing conditions. Conclusion: These findings suggest that while all methods provided high purity, the choice of protocol may significantly influence yield, RNA quality, baseline transcriptional profile and response to stimulation. This comparative study provides valuable insights to inform the choice of microglial isolation and culture method.
2024-03-28 | GSE242683 | GEO
Project description:Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance.
2016-08-05 | GSE85183 | GEO
Project description:Genomic methods are used increasingly to interrogate the individual cells that compose specific tissues. However, current methods for single cell isolation struggle to phenotypically differentiate specific cells in a heterogeneous population and rely primarily on the use of fluorescent markers. Many cellular phenotypes of interest are too complex to be measured by this approach, making it difficult to connect genotype and phenotype at the level of individual cells. Here we demonstrate that microraft arrays, which are arrays containing thousands of individual cell culture sites, can be used to select single cells based on a variety of phenotypes, such as cell surface markers, cell proliferation and drug response. We then show that a common genomic procedure, RNA-seq, can be readily adapted to the single cells isolated from these rafts. We show that data generated using microrafts and our modified RNA-seq protocol compared favorably with the Fluidigm C1. We then used microraft arrays to select pancreatic cancer cells that proliferate in spite of cytotoxic drug treatment. Our single cell RNA-seq data identified several expected and novel gene expression changes associated with early drug resistance. 120 samples including cells isolated using microrafts and the Fluidigm C1
2016-08-05 | E-GEOD-85183 | biostudies-arrayexpress
Project description:The isolation of chondrocytes from human articular cartilage for single-cell RNA sequencing requires extensive and prolonged tissue digestion at 37 °C. Modulations of the transcriptional activity likely take place during this period such that the transcriptomes of isolated human chondrocytes no longer match their original status in vivo. Here, we optimized the human chondrocyte isolation procedure to maximally preserve the in vivo transcriptome.
2022-11-15 | GSE217870 | GEO
Project description:The isolation of chondrocytes from human articular cartilage for single-cell RNA sequencing requires extensive and prolonged tissue digestion at 37 °C. Modulations of the transcriptional activity likely take place during this period such that the transcriptomes of isolated human chondrocytes no longer match their original status in vivo. Here, we optimized the human chondrocyte isolation procedure to maximally preserve the in vivo transcriptome.
2022-11-15 | GSE217869 | GEO
Project description:Methodological insight into mosquito microbiome studies: dissection and preservation
| PRJEB35477 | ENA
Project description:We analysed five different techniques to isolate phages from human adult faeces
| PRJEB30796 | ENA
Project description:Stress granules are dynamic structures, in which components belonging to the shell structure can exchange rapidly while components in the core layer may be less dynamic. The last group of proteins has been suggested to act as a scaffold to which other SG components are recruited. In a previous study, we observed that TSN does not exchange between the cytoplasm and foci upon a fluorescence recovery after photobleaching (FRAP) analysis, suggesting a role as a scaffold protein. Deletion of scaffold-like molecules has been suggest to have a high impact in membraneles organelles composition. Accordingly, and to better understand the role of TSN as a platform for SG components in plants, we characterized the SG proteome by isolation of GFP-tagged RBP47 protein from WT and double tsn1 tsn2 knock-out expressing plants.
2021-06-11 | PXD024962 | JPOST Repository