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single cell RNAseq

Gottfried Martin, David Conrad, Bertan Cakir, Günther Schlunck, Hansjürgen T Agostini
PURPOSE: Retinal vein occlusion (RVO) has been investigated in several laser-induced animal models using pigs, rabbits and rats. However, laser-induced RVO has been rarely reported in mice, despite the impressive number of available mutants, ease of handling and cost effectiveness. The aim of this study was to further assess the feasibility of a RVO mouse model for gene expression analysis and its possible use to investigate effects of hypoxia. METHODS: C57Bl/6J mice were injected with eosin Y for photo-sensitization...
2018: PloS One
Marie-Claude Carrier, Guillaume Laliberté, Eric Massé
Small regulatory RNAs (sRNAs) are ubiquitous regulatory molecules expressed in living cells. In prokaryotes, sRNAs usually bind to target mRNAs to either promote their degradation or interfere with translation initiation. Because a single sRNA can regulate a considerable number of target mRNAs, we seek to identify those targets rapidly and reliably. Here, we present a robust method based on the co-purification of target mRNAs bound to MS2-tagged sRNAs expressed in vivo. After purification of the tagged-sRNA, we use RNAseq to determine the identity of all RNA interacting partners and their enrichment level...
2018: Methods in Molecular Biology
Zhongfang Wang, Lingyan Zhu, Thi H O Nguyen, Yanmin Wan, Sneha Sant, Sergio M Quiñones-Parra, Jeremy Chase Crawford, Auda A Eltahla, Simone Rizzetto, Rowena A Bull, Chenli Qiu, Marios Koutsakos, E Bridie Clemens, Liyen Loh, Tianyue Chen, Lu Liu, Pengxing Cao, Yanqin Ren, Lukasz Kedzierski, Tom Kotsimbos, James M McCaw, Nicole L La Gruta, Stephen J Turner, Allen C Cheng, Fabio Luciani, Xiaoyan Zhang, Peter C Doherty, Paul G Thomas, Jianqing Xu, Katherine Kedzierska
Severe influenza A virus (IAV) infection is associated with immune dysfunction. Here, we show circulating CD8+ T-cell profiles from patients hospitalized with avian H7N9, seasonal IAV, and influenza vaccinees. Patient survival reflects an early, transient prevalence of highly activated CD38+ HLA-DR+ PD-1+ CD8+ T cells, whereas the prolonged persistence of this set is found in ultimately fatal cases. Single-cell T cell receptor (TCR)-αβ analyses of activated CD38+ HLA-DR+ CD8+ T cells show similar TCRαβ diversity but differential clonal expansion kinetics in surviving and fatal H7N9 patients...
February 26, 2018: Nature Communications
James R F Hockley, Toni S Taylor, Gerard Callejo, Anna L Wilbrey, Alex Gutteridge, Karsten Bach, Wendy J Winchester, David C Bulmer, Gordon McMurray, Ewan St John Smith
OBJECTIVE: Integration of nutritional, microbial and inflammatory events along the gut-brain axis can alter bowel physiology and organism behaviour. Colonic sensory neurons activate reflex pathways and give rise to conscious sensation, but the diversity and division of function within these neurons is poorly understood. The identification of signalling pathways contributing to visceral sensation is constrained by a paucity of molecular markers. Here we address this by comprehensive transcriptomic profiling and unsupervised clustering of individual mouse colonic sensory neurons...
February 26, 2018: Gut
J Stephen Dumler, Sara H Sinclair, Amol C Shetty
Eukaryotic proteome diversity exceeds that encoded within individual genes, and results in part from alternative splicing events of pre-messenger RNA. The diversity of these splicing events can shape the outcome in development and differentiation of normal tissues, and is important in pathogenic circumstances such as cancer and some heritable conditions. A role for alternative splicing of eukaryotic genes in response to viral and intracellular bacterial infections has only recently been recognized, and plays an important role in providing fitness for microbial survival, while potentially enhancing pathogenicity...
2018: Frontiers in Cellular and Infection Microbiology
Huseini Kagdi, Maria Antonietta Demontis, Juan Carlos Ramos, Graham P Taylor
Adult T-cell leukaemia/lymphoma (ATL) arises from chronic non-malignant human T lymphotropic virus type-1 (HTLV-1) infection which is characterized by high plasma pro-inflammatory cytokines whereas ATL is characterized by high plasma anti-inflammatory (IL-10) concentrations. The poor prognosis of ATL is partly ascribed to disease-associated immune suppression. ATL cells have a CD4+CCR4+CD26-CD7- immunophenotype but infected cells with this immunophenotype ('ATL-like' cells) are also present in non-malignant HTLV-1 infection...
February 14, 2018: PLoS Pathogens
Wanxin Wang, Lolita Penland, Ozgun Gokce, Derek Croote, Stephen R Quake
BACKGROUND: High-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method would greatly expand the scope of feasible multi-site collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability and fidelity are important factors to consider due to the susceptibility of single-cell RNAseq analysis to technical noise. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards...
February 13, 2018: BMC Genomics
Christel Claes, Johanna Van den Daele, Catherine M Verfaillie
Over the past decades, the importance of the immune system in a broad scope of pathologies, has drawn attention towards tissue-resident macrophages, such as microglia in the brain. To enable the study of for instance microglia, it is crucial to recreate in vitro (and in vivo) assays. However, very fast loss of tissue-specific features of primary tissue resident macrophages, including microglia, upon in vitro culture has complicated such studies. Moreover, limited knowledge of macrophage developmental pathways and the role of local 'niche factors', has hampered the generation of tissue-resident macrophages from pluripotent stem cells (PSC)...
February 2, 2018: Cellular Immunology
David Chiang, Xintong Chen, Stacie M Jones, Robert A Wood, Scott H Sicherer, A Wesley Burks, Donald Y M Leung, Charuta Agashe, Alexander Grishin, Peter Dawson, Wendy F Davidson, Leah Newman, Robert Sebra, Miriam Merad, Hugh A Sampson, Bojan Losic, M Cecilia Berin
BACKGROUND: The contribution of phenotypic variation of peanut-specific T cells to clinical allergy or tolerance to peanut is not well understood. OBJECTIVES: Our objective was to comprehensively phenotype peanut-specific T cells in the peripheral blood of individuals with and without peanut allergy (PA). METHODS: We obtained samples from PA individuals, including a cohort undergoing baseline peanut challenges for an immunotherapy trial (CoFAR6)...
January 30, 2018: Journal of Allergy and Clinical Immunology
Rajeev Sen, Igor Dolgalev, N Sumru Bayin, Adriana Heguy, Aris Tsirigos, Dimitris G Placantonakis
Single-cell RNA sequencing (sc-RNASeq) is a recently developed technique used to evaluate the transcriptome of individual cells. As opposed to conventional RNASeq in which entire populations are sequenced in bulk, sc-RNASeq can be beneficial when trying to better understand gene expression patterns in markedly heterogeneous populations of cells or when trying to identify transcriptional signatures of rare cells that may be underrepresented when using conventional bulk RNASeq. In this method, we describe the generation and analysis of cDNA libraries from single patient-derived glioblastoma cells using the C1 Fluidigm system...
2018: Methods in Molecular Biology
Marie L Landry, Ellen F Foxman
Background: Despite the high burden of respiratory infection and the importance of early and accurate diagnosis, there is no simple diagnostic test to rule in viral infection as a cause of respiratory symptoms. Methods: We performed RNASeq on human nasal epithelial cells following stimulation of the intracellular viral recognition receptor RIG-I. Next, we evaluated whether measuring identified host mRNAs and proteins from patient nasopharyngeal swabs could predict the presence of a respiratory virus in the sample...
December 21, 2017: Journal of Infectious Diseases
Carsten K Pfeffer, Riccardo Beltramo
The classification of neurons into distinct types is an ongoing effort aimed at revealing and understanding the diversity of the components of the nervous system. Recently available methods allow us to determine the gene expression pattern of individual neurons in the mammalian cerebral cortex to generate powerful categorization schemes. For a thorough understanding of neuronal diversity such genetic categorization schemes need to be combined with traditional classification parameters like position, axonal projection or response properties to sensory stimulation...
2017: Frontiers in Cellular Neuroscience
Farah Abdul-Rahman, David Gresham
RATE-seq is a 4-thiouracil (4-tU)-based method that enables the in vivo measurement of transcriptome-wide RNA degradation rates. 4-tU is an analog of uracil that is rapidly incorporated into newly synthesized RNA and facilitates the conjugation of a biotinylated molecule containing a reactive thiol group. The biotinylated RNA can then be fractionated from the unlabeled RNA with streptavidin magnetic beads. By adding 4-tU to a culture of cells growing in steady-state conditions, fractionating the labeled population of RNA at multiple time points following 4-tU addition, and quantifying the abundance of newly transcribed RNAs using RNAseq, it is possible to estimate the degradation rates of all transcripts in a single experiment...
2018: Methods in Molecular Biology
Jonathan A Griffiths, Antonio Scialdone, John C Marioni
BACKGROUND: Aneuploidies are copy number variants that affect entire chromosomes. They are seen commonly in cancer, embryonic stem cells, human embryos, and in various trisomic diseases. Aneuploidies frequently affect only a subset of cells in a sample; this is known as "mosaic" aneuploidy. A cell that harbours an aneuploidy exhibits disrupted gene expression patterns which can alter its behaviour. However, detection of aneuploidies using conventional single-cell DNA-sequencing protocols is slow and expensive...
November 25, 2017: BMC Genomics
Maud Borensztein, Ikuhiro Okamoto, Laurène Syx, Guillaume Guilbaud, Christel Picard, Katia Ancelin, Rafael Galupa, Patricia Diabangouaya, Nicolas Servant, Emmanuel Barillot, Azim Surani, Mitinori Saitou, Chong-Jian Chen, Konstantinos Anastassiadis, Edith Heard
X-chromosome inactivation is established during early development. In mice, transcriptional repression of the paternal X-chromosome (Xp) and enrichment in epigenetic marks such as H3K27me3 is achieved by the early blastocyst stage. X-chromosome inactivation is then reversed in the inner cell mass. The mechanisms underlying Xp reactivation remain enigmatic. Using in vivo single-cell approaches (allele-specific RNAseq, nascent RNA-fluorescent in situ hybridization and immunofluorescence), we show here that different genes are reactivated at different stages, with more slowly reactivated genes tending to be enriched in H3meK27...
November 3, 2017: Nature Communications
E V Poverennaya, O I Kiseleva, E A Ponomarenko, S N Naryzhny, V G Zgoda, A V Lisitsa
Current proteomic studies are generally focused on the most abundant proteoforms encoded by canonical nucleic sequences. Transcriptomic and proteomic data, accumulated in a variety of postgenome sources and coupled with state-of-art analytical technologies, allow to start the identification of aberrant (non-canonical) proteoforms. The main sources of aberrant proteoforms are alternative splicing, single nucleotide polymorphism, and post-translational modifications. The aim of this work was to estimate the heterogeneity of HepG2 proteome...
October 2017: Biomedit︠s︡inskai︠a︡ Khimii︠a︡
Mario Schiffer
Single-cell RNA-sequence (RNA-seq) is a widely used tool to study biological questions in single cells. The discussed study identified 92 genes being predominantly expressed in podocytes based on a 5-fold higher expression compared with endothelial and mesangial cells. In addition to technical pitfalls, the question that is discussed in this commentary is whether results of a single-cell RNAseq study are able to deliver expression data that truly characterize a podocyte.
November 2017: Kidney International
Gary A Clawson, Gail L Matters, Ping Xin, Christopher McGovern, Eric Wafula, Claude dePamphilis, Morgan Meckley, Joyce Wong, Luke Stewart, Christopher D'Jamoos, Naomi Altman, Yuka Imamura Kawasawa, Zhen Du, Loren Honaas, Thomas Abraham
Here we describe isolation and characterization of macrophage-tumor cell fusions (MTFs) from the blood of pancreatic ductal adenocarcinoma (PDAC) patients. The MTFs were generally aneuploidy, and immunophenotypic characterizations showed that the MTFs express markers characteristic of PDAC and stem cells, as well as M2-polarized macrophages. Single cell RNASeq analyses showed that the MTFs express many transcripts implicated in cancer progression, LINE1 retrotransposons, and very high levels of several long non-coding transcripts involved in metastasis (such as MALAT1)...
2017: PloS One
James W Wynne, Shawn Todd, Victoria Boyd, Mary Tachedjian, Reuben Klein, Brian Shiell, Megan Dearnley, Alexander J McAuley, Amanda P Woon, Anthony W Purcell, Glenn A Marsh, Michelle L Baker
Ebolavirus and Marburgvirus comprise two genera of negative-sense single-stranded RNA viruses that cause severe hemorrhagic fevers in humans. Despite considerable research efforts, the molecular events following Ebola virus (EBOV) infection are poorly understood. With the view of identifying host factors that underpin EBOV pathogenesis, we compared the transcriptomes of EBOV-infected human, pig, and bat kidney cells using a transcriptome sequencing (RNA-seq) approach. Despite a significant difference in viral transcription/replication between the cell lines, all cells responded to EBOV infection through a robust induction of extracellular growth factors...
December 1, 2017: Journal of Virology
Tessa de Bitter, Carlijn van de Water, Corina van den Heuvel, Carolien Zeelen, Astrid Eijkelenboom, Bastiaan Tops, Egbert Oosterwijk, Dimitar Kolev, Peter Mulders, Mark Ter Laan, Sanne van Lith, William Leenders
Cancer-specific metabolic alterations are of high interest as therapeutic targets. These alterations vary between tumor types, and to employ metabolic targeting to its fullest potential there is a need for robust methods that identify candidate targetable metabolic pathways in individual cancers. Currently, such methods include (13)C-tracing studies and mass spectrometry/ magnetic resonance spectroscopic imaging. Due to high cost and complexity, such studies are restricted to a research setting. We here present the validation of a novel technique of metabolic profiling, based on multiplex targeted next generation sequencing of RNA with single molecule molecular inversion probes (smMIPs), designed to measure activity of and mutations in genes that encode metabolic enzymes...
September 12, 2017: Scientific Reports
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