RESUMO
The demand for technologies that allow the study of gene expression at single cell resolution continues to increase. One such assay was launched in 2016 by the US-based company 10x Genomics Inc. Utilizing the power of the single cell on a large scale (Zheng et al. Nat Commun 8:14049, 2017)-capturing thousands of cells at once-has shaped life sciences ever since and allowed researchers to discover new insights within their respective fields of study such as oncology, neurobiology, and immunology (among others). Obtaining high-data quality is the key to being able to make these meaningful discoveries, which in turn is directly linked to the quality of the initial cell (or nuclei) suspension that is used to load the 10x Genomics Chromium Single Cell Gene Expression assay. A successful workflow relies on a cell suspension which is fully dissociated, extremely clean, and of high viability. While the workflow itself has been detailed elsewhere (De Simone et al. Methods Mol Biol 1979:87-110, 2019), in this chapter we will focus on the importance of the quality of the initial cell suspension, as well as common mistakes that can occur while running a Single Cell Gene Expression assay. The descriptions of these tips and tricks refer to the current version of the 10x Genomics User Guide (Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index). https://support10xgenomicscom/single-cell-gene-expression/index/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide-v31-chemistry-dual-index) which can be downloaded from the Support section on the 10x Genomics website (10x Genomics website. https://www10xgenomicscom). These documents and user guides are continuously improved and updated; hence, it is important to regularly check the company's website for the most recent version.
Assuntos
Genômica , Análise de Célula Única , Análise de Célula Única/métodos , Genômica/métodos , Fluxo de Trabalho , Cromo , Expressão GênicaRESUMO
BACKGROUND: The well-known Genome-Wide Association Studies (GWAS) had led to many scientific discoveries using SNP data. Even so, they were not able to explain the full heritability of complex diseases. Now, other structural variants like copy number variants or DNA inversions, either germ-line or in mosaicism events, are being studies. We present the R package affy2sv to pre-process Affymetrix CytoScan HD/750k array (also for Genome-Wide SNP 5.0/6.0 and Axiom) in structural variant studies. RESULTS: We illustrate the capabilities of affy2sv using two different complete pipelines on real data. The first one performing a GWAS and a mosaic alterations detection study, and the other detecting CNVs and performing an inversion calling. CONCLUSION: Both examples presented in the article show up how affy2sv can be used as part of more complex pipelines aimed to analyze Affymetrix SNP arrays data in genetic association studies, where different types of structural variants are considered.
Assuntos
Algoritmos , Inversão Cromossômica/genética , Variações do Número de Cópias de DNA/genética , Mosaicismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Polimorfismo de Nucleotídeo Único/genética , Software , Estudos de Associação Genética , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Hibridização in Situ Fluorescente , Inclusão em ParafinaRESUMO
BACKGROUND: Dendritic cells (DCs) are the highly specialized antigen presenting cells of the immune system that play a key role in regulating immune responses. DCs can efficiently initiate immune responses or induce tolerance. Due to this dual function, DCs are studied in the context of immunotherapy for both cancer and autoimmune diseases. Characterization of DC-specific genes, leading to better understanding of DC immunobiology, will help to guide their use in clinical settings. We previously identified DC-STAMP, a multi-membrane spanning protein preferentially expressed by DCs. DC-STAMP resides in the endoplasmic reticulum (ER) of immature DCs and translocates towards the Golgi compartment upon maturation. In this study we knocked down DC-STAMP in mouse bone marrow-derived DCs (mBMDCs) to determine its function. RESULTS: We demonstrate that DC-STAMP knock-down mBMDCs secrete less IL-6, IL-12, TNF-α and IL-10 while IL-1 production is enhanced. Moreover, LPS-matured DC-STAMP knock-down mBMDCs show impaired T cell activation potential and induction of Th1 responses in an alloreaction. CONCLUSIONS: We show that DC-STAMP plays an important role in cytokine production by mBMDCs following LPS exposure. Our results reveal a novel function of DC-STAMP in regulating DC-initiated immune responses.
