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single cell RNA sequencing

Emilia Maria Cristina Mazza, Jolanda Brummelman, Giorgia Alvisi, Alessandra Roberto, Federica De Paoli, Veronica Zanon, Federico Colombo, Mario Roederer, Enrico Lugli
Multidimensional single-cell analysis requires approaches to visualize complex data in intuitive 2D graphs. In this regard, t-distributed stochastic neighboring embedding (tSNE) is the most popular algorithm for single-cell RNA sequencing and cytometry by time-of-flight (CyTOF), but its application to polychromatic flow cytometry, including the recently developed 30-parameter platform, is still under investigation. We identified differential distribution of background values between samples, generated by either background calculation or spreading error (SE), as a major source of variability in polychromatic flow cytometry data representation by tSNE, ultimately resulting in the identification of erroneous heterogeneity among cell populations...
August 14, 2018: Cytometry. Part A: the Journal of the International Society for Analytical Cytology
Pavithra Sampath, Kadar Moideen, Uma Devi Ranganathan, Ramalingam Bethunaickan
Monocytes are critical defense components that play an important role in the primary innate immune response. The heterogeneous nature of monocytes and their ability to differentiate into either monocyte-derived macrophages or monocyte-derived dendritic cells allows them to serve as a bridge between the innate and adaptive immune responses. Current studies of monocytes based on immunofluorescence, single-cell RNA sequencing and whole mass spectrometry finger printing reveals different classification systems for monocyte subsets...
2018: Frontiers in Immunology
Urvi A Shah, Elaine Y Chung, Orsolya Giricz, Kith Pradhan, Keisuke Kataoka, Shanisha Gordon-Mitchell, Tushar D Bhagat, Yun Mai, Yongqiang Wei, Elise Ishida, Gaurav S Choudhary, Ancy Joseph, Ronald Rice, Nadege Gitego, Crystall Parrish, Matthias Bartenstein, Swati Goel, Ioannis Mantzaris, Aditi Shastri, Olga Derman, Adam Binder, Kira Gritsman, Noah Kornblum, Ira Braunschweig, Chirag Bhagat, Jeff Hall, Armin Graber, Lee Ratner, Yanhua Wang, Seishi Ogawa, Amit Verma, B Hilda Ye, Murali Janakiram
Adult T cell leukemia lymphoma (ATLL) is a rare T cell neoplasm, endemic in the Japanese, Caribbean and Latin American populations. Most North American ATLL patients are of Caribbean descent and are characterized by high rates of chemo-refractory disease and worse prognosis compared to the Japanese ATLL. To determine genomic differences between these two cohorts, we performed targeted exon sequencing on 30 North American ATLL patients and compared the results to the Japanese ATLL cases. Although the frequency of TP53 mutations was comparable, the mutation frequency in epigenetic and histone modifying genes (57%) was significantly higher whereas the mutation frequency in JAK/STAT and TCR/NF-κB pathway genes was significantly lower in our cohort...
August 13, 2018: Blood
Junil Kim, Diana E Stanescu, Kyoung Jae Won
Single-cell RNA sequencing (scRNA-seq) is a powerful tool to study heterogeneity and dynamic changes in cell populations. Clustering scRNA-seq is essential in identifying new cell types and studying their characteristics. We develop CellBIC (single Cell BImodal Clustering) to cluster scRNA-seq data based on modality in the gene expression distribution. Compared with classical bottom-up approaches that rely on a distance metric, CellBIC performs hierarchical clustering in a top-down manner. CellBIC outperformed the bottom-up hierarchical clustering approach and other recently developed clustering algorithms while maintaining the hierarchical structure of cells...
August 8, 2018: Nucleic Acids Research
Amit Zeisel, Hannah Hochgerner, Peter Lönnerberg, Anna Johnsson, Fatima Memic, Job van der Zwan, Martin Häring, Emelie Braun, Lars E Borm, Gioele La Manno, Simone Codeluppi, Alessandro Furlan, Kawai Lee, Nathan Skene, Kenneth D Harris, Jens Hjerling-Leffler, Ernest Arenas, Patrik Ernfors, Ulrika Marklund, Sten Linnarsson
The mammalian nervous system executes complex behaviors controlled by specialized, precisely positioned, and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse and were grouped by developmental anatomical units and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission, and membrane conductance...
August 9, 2018: Cell
Michael C Oldham, Anatol C Kreitzer
Single-cell RNA sequencing provides a new approach to an old problem: how to study cellular diversity in complex biological systems. Three studies-Saunders et al., Zeisel et al., and Davie et al.-deploy this technique on an unprecedented scale to reveal transcriptional patterns that distinguish cells in the nervous systems of mice and flies.
August 9, 2018: Cell
Mtakai Ngara, Mia Palmkvist, Sven Sagasser, Daisy Hjelmqvist, Åsa K Björklund, Mats Wahlgren, Johan Ankarklev, Rickard Sandberg
The malaria parasite has a complex lifecycle, including several events of differentiation and stage progression, while actively evading immunity in both its mosquito and human hosts. Important parasite gene expression and regulation during these events remain hidden in rare populations of cells. Here, we combine a capillary-based platform for cell isolation with single-cell RNA-sequencing to transcriptionally profile 165 single infected red blood cells (iRBCs) during the intra-erythrocyte developmental cycle (IDC)...
August 7, 2018: Experimental Cell Research
Luyi Tian, Shian Su, Xueyi Dong, Daniela Amann-Zalcenstein, Christine Biben, Azadeh Seidi, Douglas J Hilton, Shalin H Naik, Matthew E Ritchie
Single-cell RNA sequencing (scRNA-seq) technology allows researchers to profile the transcriptomes of thousands of cells simultaneously. Protocols that incorporate both designed and random barcodes have greatly increased the throughput of scRNA-seq, but give rise to a more complex data structure. There is a need for new tools that can handle the various barcoding strategies used by different protocols and exploit this information for quality assessment at the sample-level and provide effective visualization of these results in preparation for higher-level analyses...
August 10, 2018: PLoS Computational Biology
Garry A Luke, Martin D Ryan
Oligopeptide "2A" and "2A-like" sequences ("2As"; 18-25aa) are found in a range of RNA virus genomes controlling protein biogenesis through "recoding" of the host-cell translational apparatus. Insertion of multiple 2As within a single open reading frame (ORF) produces multiple proteins; hence, 2As have been used in a very wide range of biotechnological and biomedical applications. During translation, these 2A peptide sequences mediate a eukaryote-specific, self-"cleaving" event, termed "ribosome skipping" with very high efficiency...
August 9, 2018: Reviews in Medical Virology
Aisha A AlJanahi, Mark Danielsen, Cynthia E Dunbar
The recent development of single-cell RNA sequencing has deepened our understanding of the cell as a functional unit, providing new insights based on gene expression profiles of hundreds to hundreds of thousands of individual cells, and revealing new populations of cells with distinct gene expression profiles previously hidden within analyses of gene expression performed on bulk cell populations. However, appropriate analysis and utilization of the massive amounts of data generated from single-cell RNA sequencing experiments are challenging and require an understanding of the experimental and computational pathways taken between preparation of input cells and output of interpretable data...
September 21, 2018: Molecular Therapy. Methods & Clinical Development
Xiwen Liao, Chengkun Yang, Rui Huang, Chuangye Han, Tingdong Yu, Ketuan Huang, Xiaoguang Liu, Long Yu, Guangzhi Zhu, Hao Su, Xiangkun Wang, Wei Qin, Jianlong Deng, Xianmin Zeng, Xinping Ye, Tao Peng
BACKGROUND/AIMS: The aim of the current study was to identify potential prognostic long non-coding RNA (lncRNA) biomarkers for predicting survival in patients with hepatocellular carcinoma (HCC) using The Cancer Genome Atlas (TCGA) dataset and bioinformatics analysis. METHODS: RNA sequencing and clinical data of HCC patients from TCGA were used for prognostic association assessment by univariate Cox analysis. A prognostic signature was built using stepwise multivariable Cox analysis, and a comprehensive analysis was performed to evaluate its prognostic value...
August 9, 2018: Cellular Physiology and Biochemistry
Kei Miwata, Hirokazu Okamoto, Taku Nakashima, Daisuke Ihara, Yasushi Horimasu, Takeshi Masuda, Shintaro Miyamoto, Hiroshi Iwamoto, Kazunori Fujitaka, Hironobu Hamada, Ayumi Shibata, Takaaki Ito, Tomoyuki Okuda, Noboru Hattori
Inhalation therapy using small-interfering RNA (siRNA) is a potentially effective therapeutic strategy for lung cancer because of its high gene-silencing effects and sequence specificity. Previous studies reported that intratracheal administration of siRNA using pressurized metered dose inhalers or nebulizers could suppress tumor growth in murine lung metastatic models. Although dry powder inhalers are promising devices due to their low cost, good portability, and preservability, the anti-tumor effects of siRNA dry powder have not been elucidated...
July 17, 2018: Molecular Therapy. Nucleic Acids
Gioele La Manno, Ruslan Soldatov, Amit Zeisel, Emelie Braun, Hannah Hochgerner, Viktor Petukhov, Katja Lidschreiber, Maria E Kastriti, Peter Lönnerberg, Alessandro Furlan, Jean Fan, Lars E Borm, Zehua Liu, David van Bruggen, Jimin Guo, Xiaoling He, Roger Barker, Erik Sundström, Gonçalo Castelo-Branco, Patrick Cramer, Igor Adameyko, Sten Linnarsson, Peter V Kharchenko
RNA abundance is a powerful indicator of the state of individual cells. Single-cell RNA sequencing can reveal RNA abundance with high quantitative accuracy, sensitivity and throughput1 . However, this approach captures only a static snapshot at a point in time, posing a challenge for the analysis of time-resolved phenomena such as embryogenesis or tissue regeneration. Here we show that RNA velocity-the time derivative of the gene expression state-can be directly estimated by distinguishing between unspliced and spliced mRNAs in common single-cell RNA sequencing protocols...
August 8, 2018: Nature
Byungjin Hwang, Ji Hyun Lee, Duhee Bang
Rapid progress in the development of next-generation sequencing (NGS) technologies in recent years has provided many valuable insights into complex biological systems, ranging from cancer genomics to diverse microbial communities. NGS-based technologies for genomics, transcriptomics, and epigenomics are now increasingly focused on the characterization of individual cells. These single-cell analyses will allow researchers to uncover new and potentially unexpected biological discoveries relative to traditional profiling methods that assess bulk populations...
August 7, 2018: Experimental & Molecular Medicine
Shih-Chin Hsu, Chia-Ni Tsai, Kuan-Ying Lee, Ting-Chun Pan, Chieh-Wen Lo, Ming-Jiun Yu
The hepatitis C virus (HCV) protein NS5A is a phosphorylated protein with crucial roles in viral replication and assembly. NS5A was thought to undergo sequential phosphorylation on a series of conserved serine residues; however, the phosphorylation cascade remained obscure. Using three phosphorylation-specific antibodies, we found that phosphorylation at S232, S235, and S238 occurred in parallel in HCV-infected Huh7.5.1 cells, suggestive of intramolecular sequential NS5A phosphorylation from S232 through S235 to S238 by casein kinase I α (CKIα)...
August 8, 2018: Journal of Virology
Xiaohong Li, Eric C Rouchka, Guy N Brock, Jun Yan, Timothy E O'Toole, David A Tieri, Nigel G F Cooper
Breast cancer (BC) is increasing in incidence and resistance to treatment worldwide. The challenges in limited therapeutic options and poor survival outcomes in BC subtypes persist because of its molecular heterogeneity and resistance to standard endocrine therapy. Recently, high throughput RNA sequencing (RNA-seq) has been used to identify biomarkers of disease progression and signaling pathways that could be amenable to specific therapies according to the BC subtype. However, there is no single generally accepted pipeline for the analysis of RNA-seq data in biomarker discovery due, in part, to the needs of simultaneously satisfying constraints of sensitivity and specificity...
2018: PloS One
Dan Wang, Dejun Ma, Jingxin Han, Linghao Kong, Luyuan Li, Zhen Xi
To achieve multisite targeting-based DNA cleavage simultaneously, we designed two kinds of CRISPR RNA arrays by fusing four single sgRNAs or crRNAs with uncleavable RNA linkers (CRISPRay). CRISPRay could operate on four adjacent target sites to cleave target DNA in a collaborative manner. Two CRISPR RNA arrays demonstrated robust inactivation to the firefly luciferase gene in living cells. In vitro DNA cleavage and DNA sequencing also validated that sgRNA arrays directed SpCas9 nuclease to cut target DNA at four cleavage sites simultaneously while crRNA array-guided FnCpf1 nuclease showed target-activated, non-specific DNase activity on both target DNA and non-target DNA at random sites...
August 7, 2018: Chembiochem: a European Journal of Chemical Biology
Saki Matsumoto, Neva Caliskan, Marina V Rodnina, Asako Murata, Kazuhiko Nakatani
Programmed -1 ribosomal frameshifting (-1PRF) is a recoding mechanism to make alternative proteins from a single mRNA transcript. -1PRF is stimulated by cis-acting signals in mRNA, a seven-nucleotide slippery sequence and a downstream secondary structure element, which is often a pseudoknot. In this study we engineered the frameshifting pseudoknot from the mouse mammary tumor virus to respond to a rationally designed small molecule naphthyridine carbamate tetramer (NCTn). We demonstrate that NCTn can stabilize the pseudoknot structure in mRNA and activate -1PRF both in vitro and in human cells...
August 2, 2018: Nucleic Acids Research
Ananda L Roy, Richard Conroy, Jessica Smith, Yong Yao, Andrea C Beckel-Mitchener, James M Anderson, Elizabeth L Wilder
It has become exceedingly important to understand the precise molecular profiles of the nearly 40 trillion cells in an adult human because of their role in determining health, disease, and therapeutic outcome. The National Institutes of Health (NIH) Common Fund-supported Single Cell Analysis Program (SCAP) was designed to address this challenge. In this review, we outline the original program goals and provide a perspective on the impact of the program as a catalyst for exploration of heterogeneity of human tissues at the cellular level...
August 2018: Science Advances
Xue Li, Xianwen Meng, Cong Wei, Yincong Zhou, Hongjun Chen, He Huang, Ming Chen
Long non-coding RNAs (lncRNAs) have recently emerged as important regulators in cancer cell proliferation and metastasis. However, the role of lncRNAs in metastatic clear cell renal cell carcinoma (ccRCC) remains unclear. Here, single-cell RNA sequencing (RNA-seq) data was analyzed from primary renal cell carcinoma (pRCC) and paired metastatic renal cell carcinoma (mRCC) specimens, and characterized the expression profiles of over 10,000 genes, including 1874 lncRNAs. Further analysis revealed that lncRNAs exhibit cancer type- and tissue-specific expression across ccRCC cells...
August 6, 2018: Molecular Cancer Research: MCR
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