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https://www.readbyqxmd.com/read/28214582/structural-changes-induced-by-l50p-and-i61t-single-mutations-of-ubiquitin-affect-cell-cycle-progression-while-impairing-its-regulatory-and-degradative-functions-in-saccharomyces-cerevisiae
#1
Ankita Doshi, Mrinal Sharma, C Ratna Prabha
Posttranslational conjugation of ubiquitin to proteins either regulates their function directly or concentration through ubiquitination dependent degradation. High degree of conservation of ubiquitin's sequence implies structural and functional importance of the conserved residues. Ubiquitin gene of Saccharomyces cerevisiae was evolved in vitro by us to study the significance of conserved residues. Present study investigates the structural changes in the protein resulting from the single mutations UbS20F, UbA46S, UbL50P, UbI61T and their functional consequences in the SUB60 strain of S...
February 15, 2017: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/28212749/comparative-analysis-of-single-cell-rna-sequencing-methods
#2
Christoph Ziegenhain, Beate Vieth, Swati Parekh, Björn Reinius, Amy Guillaumet-Adkins, Martha Smets, Heinrich Leonhardt, Holger Heyn, Ines Hellmann, Wolfgang Enard
Single-cell RNA sequencing (scRNA-seq) offers new possibilities to address biological and medical questions. However, systematic comparisons of the performance of diverse scRNA-seq protocols are lacking. We generated data from 583 mouse embryonic stem cells to evaluate six prominent scRNA-seq methods: CEL-seq2, Drop-seq, MARS-seq, SCRB-seq, Smart-seq, and Smart-seq2. While Smart-seq2 detected the most genes per cell and across cells, CEL-seq2, Drop-seq, MARS-seq, and SCRB-seq quantified mRNA levels with less amplification noise due to the use of unique molecular identifiers (UMIs)...
February 16, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28212748/systematic-investigation-of-transcription-factor-activity-in-the-context-of-chromatin-using-massively-parallel-binding-and-expression-assays
#3
Michal Levo, Tali Avnit-Sagi, Maya Lotan-Pompan, Yael Kalma, Adina Weinberger, Zohar Yakhini, Eran Segal
Precise gene expression patterns are established by transcription factor (TFs) binding to regulatory sequences. While these events occur in the context of chromatin, our understanding of how TF-nucleosome interplay affects gene expression is highly limited. Here, we present an assay for high-resolution measurements of both DNA occupancy and gene expression on large-scale libraries of systematically designed regulatory sequences. Our assay reveals occupancy patterns at the single-cell level. It provides an accurate quantification of the fraction of the population bound by a nucleosome and captures distinct, even adjacent, TF binding events...
February 16, 2017: Molecular Cell
https://www.readbyqxmd.com/read/28212413/scarless-deletion-of-up-to-seven-methyl-accepting-chemotaxis-genes-with-an-optimized-method-highlights-key-function-of-chem-in-salmonella-typhimurium
#4
Stefanie Hoffmann, Christiane Schmidt, Steffi Walter, Jennifer K Bender, Roman G Gerlach
Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus...
2017: PloS One
https://www.readbyqxmd.com/read/28212192/genetic-control-of-erythropoiesis
#5
Laxminath Tumburu, Swee Lay Thein
PURPOSE OF REVIEW: The discovery of several genetic variants associated with erythroid traits and subsequent elucidation of their functional mechanisms are exemplars of the power of the new genetic and genomic technology. The present review highlights findings from recent genetic studies related to the control of erythropoiesis and dyserythropoiesis, and fetal hemoglobin, an erythroid-related trait. RECENT FINDINGS: Identification of the genetic modulators of erythropoiesis involved two approaches: genome-wide association studies (GWASs) using single nucleotide polymorphism (SNP) arrays that revealed the common genetic variants associated with erythroid phenotypes (hemoglobin, red cell count, MCV, MCH) and fetal hemoglobin; and massive parallel sequencing such as whole genome sequencing (WGS) and whole exome sequencing (WES) that led to the discovery of the rarer variants (GFI1B, SBDS, RPS19, PKLR, EPO, EPOR, KLF1, GATA1)...
February 15, 2017: Current Opinion in Hematology
https://www.readbyqxmd.com/read/28211892/high-throughput-in-situ-cell-electroporation-microsystem-for-parallel-delivery-of-single-guide-rnas-into-mammalian-cells
#6
Shengtai Bian, Yicen Zhou, Yawei Hu, Jing Cheng, Xiaofang Chen, Youchun Xu, Peng Liu
Arrayed genetic screens mediated by the CRISPR/Cas9 technology with single guide RNA (sgRNA) libraries demand a high-throughput platform capable of transfecting diverse cell types at a high efficiency in a genome-wide scale for detection and analysis of sophisticated cellular phenotypes. Here we developed a high-throughput in situ cell electroporation (HiCEP) microsystem which leveraged the superhydrophobic feature of the microwell array to achieve individually controlled conditions in each microwell and coupled an interdigital electrode array chip with the microwells in a modular-based scheme for highly efficient delivery of exogenous molecules into cells...
February 13, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28211871/expression-level-is-a-key-determinant-of-e2f1-mediated-cell-fate
#7
Igor Shats, Michael Deng, Adam Davidovich, Carolyn Zhang, Jungeun S Kwon, Dinesh Manandhar, Raluca Gordân, Guang Yao, Lingchong You
The Rb/E2F network has a critical role in regulating cell cycle progression and cell fate decisions. It is dysfunctional in virtually all human cancers, because of genetic lesions that cause overexpression of activators, inactivation of repressors, or both. Paradoxically, the downstream target of this network, E2F1, is rarely strongly overexpressed in cancer. E2F1 can induce both proliferation and apoptosis but the factors governing these critical cell fate decisions remain unclear. Previous studies have focused on qualitative mechanisms such as differential cofactors, posttranslational modification or state of other signaling pathways as modifiers of the cell fate decisions downstream of E2F1 activation...
February 17, 2017: Cell Death and Differentiation
https://www.readbyqxmd.com/read/28211676/electrochemical-genetic-profiling-of-single-cancer-cells
#8
Josep L Acero Sanchez, Hamdi Joda, Olivier Yves Henry, Beata W Solnestam, Linda Kvastad, Pelin S Akan, Joakim Lundeberg, Nadja Laddach, Dheeraj Ramakrishnan, Ian Riley, Carmen Schwind, Daniel Latta, Ciara K O' Sullivan
Recent understandings in the development and spread of cancer have lead to the realisation of novel single cell analysis platforms focused on circulating tumour cells (CTCs). A simple, rapid and inexpensive analytical platform capable of providing genetic information of these rare cells is highly desirable to support clinicians and researchers alike to either support the selection or adjustment of therapy or provide fundamental insights into cell function and cancer progression mechanisms. We report on the genetic profiling of single cancer cells, exploiting a combination of multiplex ligation-dependent probe amplification (MLPA) and electrochemical detection...
February 17, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28211111/regulation-of-nrf-operon-expression-in-pathogenic-enteric-bacteria-sequence-divergence-reveals-new-regulatory-complexity
#9
Rita E Godfrey, David J Lee, Stephen J W Busby, Douglas F Browning
The Escherichia coli K-12 nrf operon encodes a periplasmic nitrite reductase, the expression of which is driven from a single promoter, pnrf. Expression from pnrf is activated by the FNR transcription factor in response to anaerobiosis and further increased in response to nitrite by the response regulator proteins, NarL and NarP. FNR-dependent transcription is suppressed by the binding of two nucleoid associated proteins, IHF and Fis. As Fis levels increase in cells grown in rich medium, the positioning of its binding site, overlapping the promoter -10 element, ensures that pnrf is sharply repressed...
February 17, 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/28210865/the-impact-of-melanoma-genetics-on-treatment-response-and-resistance-in-clinical-and-experimental-studies
#10
M Kunz, M Hölzel
Recent attempts to characterize the melanoma mutational landscape using high-throughput sequencing technologies have identified new genes and pathways involved in the molecular pathogenesis of melanoma. Apart from mutated BRAF, NRAS, and KIT, a series of new recurrently mutated candidate genes with impact on signaling pathways have been identified such as NF1, PTEN, IDH1, RAC1, ARID2, and TP53. Under targeted treatment using BRAF and MEK1/2 inhibitors either alone or in combination, a majority of patients experience recurrences, which are due to different genetic mechanisms such as gene amplifications of BRAF or NRAS, MEK1/2 and PI3K mutations...
February 16, 2017: Cancer Metastasis Reviews
https://www.readbyqxmd.com/read/28207858/non-small-cell-lung-cancer-whole-lesion-histogram-analysis-of-the-apparent-diffusion-coefficient-for-assessment-of-tumor-grade-lymphovascular-invasion-and-pleural-invasion
#11
Naoko Tsuchiya, Mariko Doai, Katsuo Usuda, Hidetaka Uramoto, Hisao Tonami
PURPOSE: Investigating the diagnostic accuracy of histogram analyses of apparent diffusion coefficient (ADC) values for determining non-small cell lung cancer (NSCLC) tumor grades, lymphovascular invasion, and pleural invasion. MATERIALS AND METHODS: We studied 60 surgically diagnosed NSCLC patients. Diffusion-weighted imaging (DWI) was performed in the axial plane using a navigator-triggered single-shot, echo-planar imaging sequence with prospective acquisition correction...
2017: PloS One
https://www.readbyqxmd.com/read/28207771/comparison-of-whole-genome-amplification-techniques-for-human-single-cell-exome-sequencing
#12
Erik Borgström, Marta Paterlini, Jeff E Mold, Jonas Frisen, Joakim Lundeberg
BACKGROUND: Whole genome amplification (WGA) is currently a prerequisite for single cell whole genome or exome sequencing. Depending on the method used the rate of artifact formation, allelic dropout and sequence coverage over the genome may differ significantly. RESULTS: The largest difference between the evaluated protocols was observed when analyzing the target coverage and read depth distribution. These differences also had impact on the downstream variant calling...
2017: PloS One
https://www.readbyqxmd.com/read/28207000/spatial-transcriptomic-analysis-of-cryosectioned-tissue-samples-with-geo-seq
#13
Jun Chen, Shengbao Suo, Patrick Pl Tam, Jing-Dong J Han, Guangdun Peng, Naihe Jing
Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cannot be identified. Here, we describe how to use Geo-seq, a method that combines laser capture microdissection (LCM) and single-cell RNA-seq technology. The combination of these two methods enables the elucidation of cellular heterogeneity and spatial variance simultaneously...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28206693/plasmid-and-clonal-interference-during-post-horizontal-gene-transfer-evolution
#14
S Bedhomme, D Perez Pantoja, I G Bravo
Plasmids are nucleic acid molecules that can drive their own replication in a living cell. They can be transmitted horizontally and can thrive in the host cell to high copy numbers. Plasmid replication and gene expression consume cellular resources and cells carrying plasmids incur fitness costs. But many plasmids carry genes that can be beneficial under certain conditions, allowing the cell to endure in the presence of antibiotics, toxins, competitors or parasites. Horizontal transfer of plasmid-encoded genes can thus instantaneously confer differential adaptation to local or transient selection conditions...
February 16, 2017: Molecular Ecology
https://www.readbyqxmd.com/read/28205220/comprehensive-genome-profiling-of-single-sperm-cells-by-multiple-annealing-and-looping-based-amplification-cycles-and-next-generation-sequencing-from-carriers-of-robertsonian-translocation
#15
Yanwei Sha, Yankun Sha, Zhiyong Ji, Lu Ding, Qing Zhang, Honggen Ouyang, Shaobin Lin, Xu Wang, Lin Shao, Chong Shi, Ping Li, Yueqiang Song
Robertsonian translocation (RT) is a common cause for male infertility, recurrent pregnancy loss, and birth defects. Studying meiotic recombination in RT-carrier patients helps decipher the mechanism and improve the clinical management of infertility and birth defects caused by RT. Here we present a new method to study spermatogenesis on a single-gamete basis from two RT carriers. By using a combined single-cell whole-genome amplification and sequencing protocol, we comprehensively profiled the chromosomal copy number of 88 single sperms from two RT-carrier patients...
March 2017: Annals of Human Genetics
https://www.readbyqxmd.com/read/28202776/baring-cellular-souls
#16
Alex K Shalek
Coupling chemical approaches for disrupting DNA-protein complexes in intact nuclei with combinatorial barcoding enables high-throughput single-cell DNA sequencing.
February 15, 2017: Science Translational Medicine
https://www.readbyqxmd.com/read/28202731/microfluidic-droplet-platform-for-ultrahigh-throughput-single-cell-screening-of-biodiversity
#17
Stanislav S Terekhov, Ivan V Smirnov, Anastasiya V Stepanova, Tatyana V Bobik, Yuliana A Mokrushina, Natalia A Ponomarenko, Alexey A Belogurov, Maria P Rubtsova, Olga V Kartseva, Marina O Gomzikova, Alexey A Moskovtsev, Anton S Bukatin, Michael V Dubina, Elena S Kostryukova, Vladislav V Babenko, Maria T Vakhitova, Alexander I Manolov, Maja V Malakhova, Maria A Kornienko, Alexander V Tyakht, Anna A Vanyushkina, Elena N Ilina, Patrick Masson, Alexander G Gabibov, Sidney Altman
Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions...
February 15, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28199135/extreme-trait-whole-genome-sequencing-identifies-ptpro-as-a-novel-candidate-gene-in-emphysema-with-severe-airflow-obstruction
#18
Josiah E Radder, Yingze Zhang, Alyssa D Gregory, Shibing Yu, Neil J Kelly, Joseph K Leader, Naftali Kaminski, Frank C Sciurba, Steven D Shapiro
RATIONALE: Genetic association studies in chronic obstructive pulmonary disease (COPD) have primarily tested for association with common variants, the results of which explain only a portion of disease heritability. As rare variation is also likely to contribute to susceptibility, we employed whole genome sequencing (WGS) of subjects with clinically extreme phenotypes to identify genomic regions enriched for rare variation contributing to COPD susceptibility. OBJECTIVE: Identify regions of rare genetic variation contributing to emphysema with severe airflow obstruction Methods: We identified heavy smokers that were resistant (n=65) or susceptible (n=64) to emphysema with severe airflow obstruction in the Pittsburgh SCCOR cohort...
February 15, 2017: American Journal of Respiratory and Critical Care Medicine
https://www.readbyqxmd.com/read/28198391/mcm5-a-new-actor-in-the-link-between-dna-replication-and-meier-gorlin-syndrome
#19
Annalisa Vetro, Salvatore Savasta, Annalisa Russo Raucci, Cristina Cerqua, Geppo Sartori, Ivan Limongelli, Antonella Forlino, Silvia Maruelli, Paola Perucca, Debora Vergani, Giuliano Mazzini, Andrea Mattevi, Lucia Anna Stivala, Leonardo Salviati, Orsetta Zuffardi
Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5...
February 15, 2017: European Journal of Human Genetics: EJHG
https://www.readbyqxmd.com/read/28198357/cgcorrect-a-method-to-correct-for-confounding-cell-cell-variation-due-to-cell-growth-in-single-cell-transcriptomics
#20
Thomas Blasi, Florian Buettner, Michael Strasser, Carsten Marr, Fabian Theis
MOTIVATION: Accessing gene expression at the single cell level has unraveled often large heterogeneity among seemingly homogeneous cells, which remained obscured in traditional population based approaches. The computational analysis of single-cell transcriptomics data, however, still imposes unresolved challenges with respect to normalization, visualization and modeling the data. One such issue are differences in cell size, which introduce additional variability into the data, for which appropriate normalization techniques are needed...
February 15, 2017: Physical Biology
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