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single-cell sequencing

Shiqi Luo, Feng He, Junjie Luo, Shengqian Dou, Yirong Wang, Annan Guo, Jian Lu
Transfer RNA-derived small RNAs (tsRNAs) are an emerging class of small RNAs, yet their regulatory roles have not been well understood. Here we studied the molecular mechanisms and consequences of tsRNA-mediated regulation in Drosophila. By analyzing 495 public small RNA libraries, we demonstrate that most tsRNAs are conserved, prevalent and abundant in Drosophila. By carrying out mRNA sequencing and ribosome profiling of S2 cells transfected with single-stranded tsRNA mimics and mocks, we show that tsRNAs recognize target mRNAs through conserved complementary sequence matching and suppress target genes by translational inhibition...
March 14, 2018: Nucleic Acids Research
Charles Cole, Ashley Byrne, Anna E Beaudin, E Camilla Forsberg, Christopher Vollmers
RNA-sequencing (RNA-seq) is a powerful technique to investigate and quantify entire transcriptomes. Recent advances in the field have made it possible to explore the transcriptomes of single cells. However, most widely used RNA-seq protocols fail to provide crucial information regarding transcription start sites. Here we present a protocol, Tn5Prime, that takes advantage of the Tn5 transposase-based Smart-seq2 protocol to create RNA-seq libraries that capture the 5' end of transcripts. The Tn5Prime method dramatically streamlines the 5' capture process and is both cost effective and reliable...
March 14, 2018: Nucleic Acids Research
Peter Vegh, Muzlifah Haniffa
Application of single-cell genomics technologies has revolutionized our approach to study the immune system. Unravelling the functional diversity of immune cells and their coordinated response is key to understanding immunity. Single-cell transcriptomics technologies provide high-dimensional assessment of the transcriptional states of immune cells and have been successfully applied to discover new immune cell types, reveal haematopoietic lineages, identify gene modules dictating immune responses and investigate lymphocyte antigen receptor diversity...
March 14, 2018: Briefings in Functional Genomics
Amporn Suphatrakul, Thaneeya Duangchinda, Natapong Jupatanakul, Kanjanawadee Prasittisa, Suppachoke Onnome, Jutharat Pengon, Bunpote Siridechadilok
Reporter virus is a versatile tool to visualize and to analyze virus infections. However, for flaviviruses, it is difficult to maintain the inserted reporter genes on the viral genome, limiting its use in several studies that require homogeneous virus particles and several rounds of virus replication. Here, we showed that flanking inserted GFP genes on both sides with ribosome-skipping 2A sequences improved the stability and the consistency of their fluorescent signals for dengue-virus-serotype 2 (DENV2) reporter viruses...
2018: PloS One
Davina Gale, Andrew R J Lawson, Karen Howarth, Mikidache Madi, Bradley Durham, Sarah Smalley, John Calaway, Shannon Blais, Greg Jones, James Clark, Peter Dimitrov, Michelle Pugh, Samuel Woodhouse, Michael Epstein, Ana Fernandez-Gonzalez, Alexandra S Whale, Jim F Huggett, Carole A Foy, Gerwyn M Jones, Hadas Raveh-Amit, Karin Schmitt, Alison Devonshire, Emma Green, Tim Forshew, Vincent Plagnol, Nitzan Rosenfeld
INTRODUCTION: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes...
2018: PloS One
Renata Gruszka, Magdalena Zakrzewska
The fundamental function of ribonucleic acids is to transfer genetic information from DNA to protein during translation process, however, this is not the only way connecting active RNA sequences with essential biological processes. Up until now, many RNA subclasses of different size, structure, and biological function were identified. Among them, there are non-coding single-stranded microRNAs (miRNAs). This subclass comprises RNAs of 19-25 nucleotides in length that modulate the activity of well-defined coding RNAs and play a crucial role in many physiological and pathological processes...
March 16, 2018: International Journal of Molecular Sciences
Qingwang Xue, Chunxue Liu, Xia Li, Li Dai, Huaisheng Wang
Various fluorescent sensing systems for miRNAs detection have been developed. But they mostly contain enzymatic amplification reactions and label procedures. The strict reaction conditions of tool enzymes and the high cost of labeling limit their potential applications, especially in complex biological matrix. Here we have addressed the difficult problems and report a strategy for label-free fluorescent DNA dendrimers based on enzyme-free nonlinear hybridization chain reaction (HCR)-mediated multiple G-quadruplex for simple, sensitive and selective detection of miRNAs with low-background signal...
March 16, 2018: Bioconjugate Chemistry
Te-Sha Tsai, Sonika Tyagi, Justin C St John
STUDY QUESTION: What are the molecular differences between mitochondrial DNA (mtDNA)-deficient and mtDNA-normal oocytes and how does mitochondrial supplementation alter these? SUMMARY ANSWER: Changes to DNA methylation in a 5' cytosine-phosphate-guanine 3' (CpG) island in the mtDNA-specific replication factor (DNA polymerase gamma (POLG)) of mtDNA-deficient oocytes mediates an increase in mtDNA copy number by the 2-cell stage that positively modulates the expression of nuclear genes, which affect cellular and metabolic processes, following autologous mitochondrial supplementation...
March 13, 2018: Human Reproduction
Alexander B Rosenberg, Charles M Roco, Richard A Muscat, Anna Kuchina, Paul Sample, Zizhen Yao, Lucas Gray, David J Peeler, Sumit Mukherjee, Wei Chen, Suzie H Pun, Drew L Sellers, Bosiljka Tasic, Georg Seelig
To facilitate scalable profiling of single cells, we developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), a single-cell RNA-seq (scRNA-seq) method that labels the cellular origin of RNA through combinatorial barcoding. SPLiT-seq is compatible with fixed cells or nuclei, allows efficient sample multiplexing and requires no customized equipment. We used SPLiT-seq to analyze 156,049 single-nucleus transcriptomes from postnatal day 2 and 11 mouse brains and spinal cords. Over 100 cell types were identified, with gene expression patterns corresponding to cellular function, regional specificity, and stage of differentiation...
March 15, 2018: Science
Shiwei Zheng, Efthymia Papalexi, Andrew Butler, William Stephenson, Rahul Satija
Hematopoietic stem cells (HSCs) give rise to diverse cell types in the blood system, yet our molecular understanding of the early trajectories that generate this enormous diversity in humans remains incomplete. Here, we leverage Drop-seq, a massively parallel single-cell RNA sequencing (scRNA-seq) approach, to individually profile 20,000 progenitor cells from human cord blood, without prior enrichment or depletion for individual lineages based on surface markers. Our data reveal a transcriptional compendium of progenitor states in human cord blood, representing four committed lineages downstream from HSC, alongside the transcriptional dynamics underlying fate commitment...
March 15, 2018: Molecular Systems Biology
Holger Winkels, Erik Ehinger, Melanie Vassallo, Konrad Buscher, Huy Dinh, Kouji Kobiyama, Anouk Hamers, Clément Cochain, Ehsan Vafadarnejad, Antoine-Emmanuel Saliba, Alma Zernecke, Akula Pramod, Amlan Ghosh, Nathaly Anto Michel, Natalie Hoppe, Ingo Hilgendorf, Andreas Zirlik, Catherine Hedrick, Klaus Ley, Dennis Wolf
<u>Rationale:</u> Atherosclerosis is a chronic inflammatory disease that is driven by the interplay of pro- and anti-inflammatory leukocytes in the aorta. Yet, the phenotypic and transcriptional diversity of aortic leukocytes is only poorly understood. <u>Objective:</u> We characterized leukocytes from healthy and atherosclerotic mouse aortas in-depth by single cell RNA-sequencing (scRNAseq) and mass cytometry (CyTOF) to define an atlas of the immune cell landscape in atherosclerosis...
March 15, 2018: Circulation Research
Clément Cochain, Ehsan Vafadarnejad, Panagiota Arampatzi, Pelisek Jaroslav, Holger Winkels, Klaus Ley, Dennis Wolf, Antoine-Emmanuel Saliba, Alma Zernecke
<u>Rationale:</u> It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they ha ve been defined by the expression of a restricted number of markers. <u>Objective:</u> We have applied single-cell RNA-seq as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis. <u>Methods and Results:</u> We performed single-cell RNA sequencing of total aortic CD45+ cells extracted from the non-diseased (chow fed) and atherosclerotic (11 weeks of high fat diet) aorta of Ldlr-/- mice...
March 15, 2018: Circulation Research
Sofie Ellebæk Pollmann, Valerie S Calvert, Shruti Rao, Simina M Boca, Subha Madhavan, Ivan D Horak, Andreas Kjaer, Emanuel F Petricoin, Michael Kragh, Thomas Tuxen Poulsen
Failure of clinical trials due to development of resistance to MET-targeting therapeutic agents is an emerging problem. Mechanisms of acquired resistance to MET tyrosine kinase inhibitors are well described, whereas characterization of mechanisms of resistance toward MET-targeting antibodies is limited. This study investigated mechanisms underlying in vivo resistance to two antibody therapeutics currently in clinical development: an analogue of the MET-targeting antibody emibetuzumab and Sym015, a mixture of two antibodies targeting non-overlapping epitopes of MET...
March 15, 2018: Molecular Cancer Therapeutics
Qing Mao, Robert Chin, Weiwei Xie, Yuqing Deng, Wenwei Zhang, Huixin Xu, Rebecca Yu Zhang, Quan Shi, Erin E Peters, Natali Gulbahce, Zhenyu Li, Fang Chen, Radoje Drmanac, Brock A Peters
BACKGROUND: Amniocentesis is a common procedure, the primary purpose of which is to collect cells from the fetus to allow testing for abnormal chromosomes, altered chromosomal copy number, or a small number of genes that have small single- to multibase defects. Here we demonstrate the feasibility of generating an accurate whole-genome sequence of a fetus from either the cellular or cell-free DNA (cfDNA) of an amniotic sample. METHODS: cfDNA and DNA isolated from the cell pellet of 31 amniocenteses were sequenced to approximately 50× genome coverage by use of the Complete Genomics nanoarray platform...
March 15, 2018: Clinical Chemistry
Suhyung Cho, Donghui Choe, Eunju Lee, Sun Chang Kim, Bernhard Ø Palsson, Byung-Kwan Cho
Along with functional advances in the use of CRISPR/Cas9 for genome editing, endonuclease-deficient Cas9 (dCas9) has provided a versatile molecular tool for exploring gene functions. In principle, differences in cell phenotypes that result from the RNA-guided modulation of transcription levels by dCas9 are critical for inferring with gene function; however, the effect of intracellular dCas9 expression on bacterial morphology has not been systematically elucidated. Here, we observed unexpected morphological changes in Escherichia coli mediated by dCas9, which were then characterized using RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq)...
March 15, 2018: ACS Synthetic Biology
Marian L Miller, Aleksey Porollo, Susan Wert
Alveolar type II cells from seven mammalian species were examined for a protein in the rough endoplasmic reticulum (RER), which showed a multi-layered, repeating motif. Each motif, 100nm in width, comprised two parallel outer dense layers, a less dense central layer, and often 1-3 faint layers on either side of the latter. Outer layers showed periodicities at 3-4 densities/100nm of width, while layers on either side of the central layer showed 5-7 densities/100nm of width. RER membranes were ribosome-free when parallel to these layers, but showed 4 ribosomes per motif at the growing ends: one ribosome at each outer dense layer, and one on either side of the less dense central layer...
March 15, 2018: Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology
Eva Vargas, Eloy Povedano, Víctor Ruiz-Valdepeñas Montiel, Rebeca M Torrente-Rodríguez, Mohamed Zouari, Juan José Montoya, Noureddine Raouafi, Susana Campuzano, José M Pingarrón
This work reports an amperometric biosensor for the determination of miRNA-21, a relevant oncogene. The methodology involves a competitive DNA-target miRNA hybridization assay performed on the surface of magnetic microbeads (MBs) and amperometric transduction at screen-printed carbon electrodes (SPCEs). The target miRNA competes with a synthetic fluorescein isothiocyanate (FITC)-modified miRNA with an identical sequence for hybridization with a biotinylated and complementary DNA probe (b-Cp) immobilized on the surface of streptavidin-modified MBs (b-Cp-MBs)...
March 15, 2018: Sensors
Fatemeh Faraji, Nader Tajik, Mahdi Behdani, Mohammad Ali Shokrgozar, Amir Hassan Zarnani, Fatemeh Shahhosseini, Mahdi Habibi-Anbouhi
CD22 is a B-cell-specific trans-membrane glycoprotein which is found on the surface of the most B-cells and modulates their function, survival, and apoptosis. Recently, targeting this cell surface biomarker in B-cell malignancies and disorders has attracted a lot of attention. The variable domain of camelid single chain antibodies (VHH, Nanobody) is a form of antibodies with novel properties including small size (15-17 kDa), thermal and chemical stability, high affinity and homology to human antibody sequences...
March 15, 2018: Biotechnology and Applied Biochemistry
Kelly Kyker-Snowman, Bracha Erlanger Avigdor, Mansoor Nasim, Ashley Cimino-Mathews, Sarah J Wheelan, Pedram Argani, Ben Ho Park
BACKGROUND/PURPOSE: Tumor heterogeneity is a now well-recognized phenomenon that can affect the classification, prognosis and treatment of human cancers. Heterogeneity is often described in primary breast cancers based upon histologic subtypes, hormone- and HER2-receptor status, and immunolabeling for various markers, which can be seen within a single tumor as mixed cellular populations, or as separate discrete foci. EXPERIMENTAL DESIGN/METHODS: Here, we present a case report of a patient's primary breast cancer that had two separate but adjacent histologic components, one that was estrogen receptor (ER) positive, and the other ER negative...
March 15, 2018: Breast Cancer Research and Treatment
Cosimo Cumbo, Luciana Impera, Crescenzio Francesco Minervini, Paola Orsini, Luisa Anelli, Antonella Zagaria, Nicoletta Coccaro, Giuseppina Tota, Angela Minervini, Paola Casieri, Claudia Brunetti, Antonella Russo Rossi, Elisa Parciante, Giorgina Specchia, Francesco Albano
For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology...
February 16, 2018: Oncotarget
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