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single cell RNAseq

James C Chen, Angela M Christiano
Mammalian skin is a complex and heterogeneous tissue with several distinct compartments and stem cell populations. Joost et al. (2016) now use single-cell RNaseq to comprehensively reconstruct this complexity, revealing spatial and pseudotemporal differences between transcriptional programs in distinct compartments and a common basal program in skin stem cell populations.
October 6, 2016: Cell Stem Cell
Jonathan W Nelson, Jiri Sklenar, Anthony P Barnes, Jessica Minnier
: Transcriptional profiling using RNA sequencing (RNAseq) has emerged as a powerful methodology to quantify global gene expression patterns in various contexts from single cells to whole tissues. The tremendous amount of data generated by this profiling technology presents a daunting challenge in terms of effectively visualizing and interpreting results. Convenient and intuitive data interfaces are critical for researchers to easily upload, analyze, and visualize their RNAseq data. We designed the START (Shiny Transcriptome Analysis Resource Tool) App with these requirements in mind...
September 30, 2016: Bioinformatics
Dorrelyn P Martin, Jharna Miya, Julie W Reeser, Sameek Roychowdhury
RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods...
2016: Journal of Visualized Experiments: JoVE
Csaba Földy, Spyros Darmanis, Jason Aoto, Robert C Malenka, Stephen R Quake, Thomas C Südhof
In brain, signaling mediated by cell adhesion molecules defines the identity and functional properties of synapses. The specificity of presynaptic and postsynaptic interactions that is presumably mediated by cell adhesion molecules suggests that there exists a logic that could explain neuronal connectivity at the molecular level. Despite its importance, however, the nature of such logic is poorly understood, and even basic parameters, such as the number, identity, and single-cell expression profiles of candidate synaptic cell adhesion molecules, are not known...
August 30, 2016: Proceedings of the National Academy of Sciences of the United States of America
Yurong Xin, Haruka Okamoto, Jinrang Kim, Min Ni, Christina Adler, Katie Cavino, Erqian Na, Andrew J Murphy, George D Yancopoulos, Calvin Lin, Jesper Gromada
Aging improves pancreatic β-cell function in mice. This is a surprising finding because aging is typically associated with functional decline. We performed single-cell RNA sequencing of β-cells from 3- and 26-month-old mice to explore how changes in gene expression contribute to improved function with age. The old mice were healthy and had reduced blood glucose levels and increased β-cell mass, which correlated to their body weight. β-Cells from young and old mice had similar transcriptome profiles. In fact, only 193 genes (0...
September 2016: Endocrinology
David Redmond, Asaf Poran, Olivier Elemento
Accurate characterization of the repertoire of the T-cell receptor (TCR) alpha and beta chains is critical to understanding adaptive immunity. Such characterization has many applications across such fields as vaccine development and response, clone-tracking in cancer, and immunotherapy. Here we present a new methodology called single-cell TCRseq (scTCRseq) for the identification and assembly of full-length rearranged V(D)J T-cell receptor sequences from paired-end single-cell RNA sequencing reads. The method allows accurate identification of the V(D)J rearrangements for each individual T-cell and has the novel ability to recover paired alpha and beta segments...
2016: Genome Medicine
Soren Impey, Carl Pelz, Amanuel Tafessu, Tessa Marzulla, Mitchell S Turker, Jacob Raber
BACKGROUND: Proton irradiation poses a potential hazard to astronauts during and following a mission, with post-mitotic cells at most risk because they cannot dilute resultant epigenetic changes via cell division. Persistent epigenetic changes that result from environmental exposures include gains or losses of DNA methylation of cytosine, which can impact gene expression. In the present study, we compared the long-term epigenetic effects of whole body proton irradiation in the mouse hippocampus and left ventricle...
2016: BMC Genomics
Nachiket Shembekar, Chawaree Chaipan, Ramesh Utharala, Christoph A Merten
Droplet-based microfluidics enables assays to be carried out at very high throughput (up to thousands of samples per second) and enables researchers to work with very limited material, such as primary cells, patient's biopsies or expensive reagents. An additional strength of the technology is the possibility to perform large-scale genotypic or phenotypic screens at the single-cell level. Here we critically review the latest developments in antibody screening, drug discovery and highly multiplexed genomic applications such as targeted genetic workflows, single-cell RNAseq and single-cell ChIPseq...
April 21, 2016: Lab on a Chip
Pan Zhang, David C Samuels, Jing Wang, Shilin Zhao, Yu Shyr, Yan Guo
It has been shown that heteroplasmic mitochondrial DNA variants can be tissue specific. However, whether mitochondrial DNA variants are specific by blood cell types has not been investigated. Motivated by this question and using mitochondria sequences extracted from RNAseq data from six distinct blood cell types (neutrophil, monocyte, myeloid dendritic, natural killer, T and B), we thoroughly compared SNPs and heteroplasmies among these cell types. Each cell type from each subject was sequenced at four time points used as biological replicates...
May 2016: Mitochondrion
S Postel-Vinay, Y Boursin, C Massard, A Hollebecque, E Ileana, M Chiron, J Jung, J S Lee, Z Balogh, J Adam, P Vielh, E Angevin, L Lacroix, J-C Soria
BACKGROUND: Molecular tumour profiling technologies have become increasingly important in the era of precision medicine, but their routine use is limited by their accessibility, cost, and tumour material availability. It is therefore crucial to assess their relative added value to optimize the sequence and combination of such technologies. PATIENTS AND METHODS: Within the MOSCATO-01 trial, we investigated the added value of whole exome sequencing (WES) in patients that did not present any molecular abnormality on array comparative genomic hybridization (aCGH) and targeted gene panel sequencing (TGPS) using cancer specific panels...
February 2016: Annals of Oncology: Official Journal of the European Society for Medical Oncology
Long M G Bui, Stephen P Kidd
A key to persistent and recurrent Staphylococcus aureus infections is its ability to adapt to diverse and toxic conditions. This ability includes a switch into a biofilm or to the quasi-dormant Small Colony Variant (SCV). The development and molecular attributes of SCVs have been difficult to study due to their rapid reversion to their parental cell-type. We recently described the unique induction of a matrix-embedded and stable SCV cell-type in a clinical S. aureus strain (WCH-SK2) by growing the cells with limiting conditions for a prolonged timeframe...
December 2015: Infection, Genetics and Evolution
Mariacristina Di Marco, Annalisa Astolfi, Elisa Grassi, Silvia Vecchiarelli, Marina Macchini, Valentina Indio, Riccardo Casadei, Claudio Ricci, Marielda D'Ambra, Giovanni Taffurelli, Carla Serra, Giorgio Ercolani, Donatella Santini, Antonia D'Errico, Antonio Daniele Pinna, Francesco Minni, Sandra Durante, Laura Raffaella Martella, Guido Biasco
The aim of the current study was to implement whole transcriptome massively parallel sequencing (RNASeq) and copy number analysis to investigate the molecular biology of pancreatic ductal adenocarcinoma (PDAC). Samples from 16 patients with PDAC were collected by ultrasound‑guided biopsy or from surgical specimens for DNA and RNA extraction. All samples were analyzed by RNASeq performed at 75x2 base pairs on a HiScanSQ Illumina platform. Single‑nucleotide variants (SNVs) were detected with SNVMix and filtered on dbSNP, 1000 Genomes and Cosmic...
November 2015: Molecular Medicine Reports
Troy A Hackett, Yan Guo, Amanda Clause, Nicholas J Hackett, Krassimira Garbett, Pan Zhang, Daniel B Polley, Karoly Mirnics
BACKGROUND: The maturation of the brain involves the coordinated expression of thousands of genes, proteins and regulatory elements over time. In sensory pathways, gene expression profiles are modified by age and sensory experience in a manner that differs between brain regions and cell types. In the auditory system of altricial animals, neuronal activity increases markedly after the opening of the ear canals, initiating events that culminate in the maturation of auditory circuitry in the brain...
2015: BMC Genomics
Pan Zhang, David C Samuels, Brian Lehmann, Thomas Stricker, Jennifer Pietenpol, Yu Shyr, Yan Guo
The rapid progress in high-throughput sequencing has significantly enriched our capacity for studying the mitochondrial DNA (mtDNA). In addition to performing specific mitochondrial targeted sequencing, an increasingly popular alternative approach is using the off-target reads from exome sequencing to infer mtDNA variants, including single nucleotide polymorphisms (SNPs) and heteroplasmy. However, the effectiveness and practicality of this approach have not been tested. Recently, RNAseq data have also been suggested as a good source for alternative data mining, but whether mitochondrial variants can be detected from RNAseq data has not been validated...
March 2016: Briefings in Bioinformatics
Scott Powers, Matt DeJongh, Aaron A Best, Nathan L Tintle
BACKGROUND: Rapid growth in the availability of genome-wide transcript abundance levels through gene expression microarrays and RNAseq promises to provide deep biological insights into the complex, genome-wide transcriptional behavior of single-celled organisms. However, this promise has not yet been fully realized. RESULTS: We find that computation of pairwise gene associations (correlation; mutual information) across a set of 2782 total genome-wide expression samples from six diverse bacteria produces unexpectedly large variation in estimates of pairwise gene association-regardless of the metric used, the organism under study, or the number and source of the samples...
2015: Frontiers in Microbiology
Maria Carolina Viana, Albert Nobre Menezes, Miguel Angelo M Moreira, Alcides Pissinatti, Héctor N Seuánez
BACKGROUND: The methyl-CpG Binding Protein two gene (MECP2) encodes a multifunctional protein comprising two isoforms involved in nuclear organization and regulation of splicing and mRNA template activity. This gene is normally expressed in all tissues, with a higher expression level in the brain during neuronal maturation. Loss of MECP2 function is the primary cause of Rett syndrome (RTT) in humans, a dominant, X-linked disorder dramatically affecting neural and motor development. RESULTS: We investigated the molecular evolution of MECP2 in several primate taxa including 36 species in 16 genera of neotropical (platyrrhine) primates...
2015: BMC Genetics
Jenette Creaney, Shaokang Ma, Sophie A Sneddon, Michelle R Tourigny, Ian M Dick, Justine S Leon, Andrea Khong, Scott A Fisher, Richard A Lake, W Joost Lesterhuis, Anna K Nowak, Shay Leary, Mark W Watson, Bruce W Robinson
A key to improving cancer immunotherapy will be the identification of tumor-specific "neoantigens" that arise from mutations and augment the resultant host immune response. In this study we identified single nucleotide variants (SNVs) by RNA sequencing of asbestos-induced murine mesothelioma cell lines AB1 and AB1-HA. Using the NetMHCpan 2.8 algorithm, the theoretical binding affinity of predicted peptides arising from high-confidence, exonic, non-synonymous SNVs was determined for the BALB/c strain. The immunoreactivity to 20 candidate mutation-carrying peptides of increased affinity and the corresponding wild-type peptides was determined using interferon-γ ELISPOT assays and lymphoid organs of non-manipulated tumor-bearing mice...
July 2015: Oncoimmunology
Álvaro Díaz, Cecilia Fernández, Álvaro Pittini, Paula I Seoane, Judith E Allen, Cecilia Casaravilla
The laminated layer is the unique mucin-based extracellular matrix that protects Echinococcus larvae, and thus to an important extent, shapes host-parasite relationships in the larval echinococcoses. In 2011, we published twin reviews summarizing what was known about this structure. Since then, important advances have been made. Complete genomes and some RNAseq data are now available for E. multilocularis and E. granulosus, leading to the inference that the E. multilocularis LL is probably formed by a single type of mucin backbone, while a second apomucin subfamily additionally contributes to the E...
November 2015: Experimental Parasitology
Jason A Morrison, Andrew C Box, Mary C McKinney, Rebecca McLennan, Paul M Kulesa
BACKGROUND: Single cell gene profiling has been successfully applied to cultured cells. However, isolation and preservation of a cell's native gene expression state from an intact embryo remain problematic. RESULTS: Here, we present a strategy for in vivo single cell profiling that optimizes cell identification, isolation and amplification of nucleic acids with nominal bias and sufficient material detection. We first tested several photoconvertible fluorescent proteins to selectively mark a cell(s) of interest in living chick embryos then accurately identify and isolate the same cell(s) in fixed tissue slices...
June 2015: Developmental Dynamics: An Official Publication of the American Association of Anatomists
Iakes Ezkurdia, Jose Manuel Rodriguez, Enrique Carrillo-de Santa Pau, Jesús Vázquez, Alfonso Valencia, Michael L Tress
Although eukaryotic cells express a wide range of alternatively spliced transcripts, it is not clear whether genes tend to express a range of transcripts simultaneously across cells, or produce dominant isoforms in a manner that is either tissue-specific or regardless of tissue. To date, large-scale investigations into the pattern of transcript expression across distinct tissues have produced contradictory results. Here, we attempt to determine whether genes express a dominant splice variant at the protein level...
April 3, 2015: Journal of Proteome Research
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