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Nature Methods

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https://www.readbyqxmd.com/read/28099430/pooled-crispr-screening-with-single-cell-transcriptome-readout
#1
Paul Datlinger, André F Rendeiro, Christian Schmidl, Thomas Krausgruber, Peter Traxler, Johanna Klughammer, Linda C Schuster, Amelie Kuchler, Donat Alpar, Christoph Bock
CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells...
January 18, 2017: Nature Methods
https://www.readbyqxmd.com/read/28092692/smile-seq-identifies-binding-motifs-of-single-and-dimeric-transcription-factors
#2
Alina Isakova, Romain Groux, Michael Imbeault, Pernille Rainer, Daniel Alpern, Riccardo Dainese, Giovanna Ambrosini, Didier Trono, Philipp Bucher, Bart Deplancke
Resolving the DNA-binding specificities of transcription factors (TFs) is of critical value for understanding gene regulation. Here, we present a novel, semiautomated protein-DNA interaction characterization technology, selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq). SMiLE-seq is neither limited by DNA bait length nor biased toward strong affinity binders; it probes the DNA-binding properties of TFs over a wide affinity range in a fast and cost-effective fashion. We validated SMiLE-seq by analyzing 58 full-length human, mouse, and Drosophila TFs from distinct structural classes...
January 16, 2017: Nature Methods
https://www.readbyqxmd.com/read/28092691/effective-detection-of-variation-in-single-cell-transcriptomes-using-matq-seq
#3
Kuanwei Sheng, Wenjian Cao, Yichi Niu, Qing Deng, Chenghang Zong
The quantification of transcriptional variation in single cells, particularly within the same cell population, is currently limited by the low sensitivity and high technical noise of single-cell RNA-seq assays. We report multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq), a highly sensitive and quantitative method for single-cell sequencing of total RNA. By systematically determining technical noise, we show that MATQ-seq captures genuine biological variation between whole transcriptomes of single cells...
January 16, 2017: Nature Methods
https://www.readbyqxmd.com/read/28092690/post-translational-selective-intracellular-silencing-of-acetylated-proteins-with-de-novo-selected-intrabodies
#4
Michele Chirichella, Simonetta Lisi, Marco Fantini, Martina Goracci, Mariantonietta Calvello, Rossella Brandi, Ivan Arisi, Mara D'Onofrio, Cristina Di Primio, Antonino Cattaneo
The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells...
January 16, 2017: Nature Methods
https://www.readbyqxmd.com/read/28068317/cryogenic-optical-localization-provides-3d-protein-structure-data-with-angstrom-resolution
#5
Siegfried Weisenburger, Daniel Boening, Benjamin Schomburg, Karin Giller, Stefan Becker, Christian Griesinger, Vahid Sandoghdar
We introduce Cryogenic Optical Localization in 3D (COLD), a method to localize multiple fluorescent sites within a single small protein with Angstrom resolution. We demonstrate COLD by determining the conformational state of the cytosolic Per-ARNT-Sim domain from the histidine kinase CitA of Geobacillus thermodenitrificans and resolving the four biotin sites of streptavidin. COLD provides quantitative 3D information about small- to medium-sized biomolecules on the Angstrom scale and complements other techniques in structural biology...
January 9, 2017: Nature Methods
https://www.readbyqxmd.com/read/28068316/scalable-whole-genome-single-cell-library-preparation-without-preamplification
#6
Hans Zahn, Adi Steif, Emma Laks, Peter Eirew, Michael VanInsberghe, Sohrab P Shah, Samuel Aparicio, Carl L Hansen
Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations...
January 9, 2017: Nature Methods
https://www.readbyqxmd.com/read/28068315/hyperspectral-phasor-analysis-enables-multiplexed-5d-in-vivo-imaging
#7
Francesco Cutrale, Vikas Trivedi, Le A Trinh, Chi-Li Chiu, John M Choi, Marcela S Artiga, Scott E Fraser
Time-lapse imaging of multiple labels is challenging for biological imaging as noise, photobleaching and phototoxicity compromise signal quality, while throughput can be limited by processing time. Here, we report software called Hyper-Spectral Phasors (HySP) for denoising and unmixing multiple spectrally overlapping fluorophores in a low signal-to-noise regime with fast analysis. We show that HySP enables unmixing of seven signals in time-lapse imaging of living zebrafish embryos.
January 9, 2017: Nature Methods
https://www.readbyqxmd.com/read/28024160/resa-identifies-mrna-regulatory-sequences-at-high-resolution
#8
Valeria Yartseva, Carter M Takacs, Charles E Vejnar, Miler T Lee, Antonio J Giraldez
Gene expression is extensively regulated at the levels of mRNA stability, localization and translation. However, decoding functional RNA-regulatory features remains a limitation to understanding post-transcriptional regulation in vivo. Here, we developed RNA-element selection assay (RESA), a method that selects RNA elements on the basis of their activity in vivo and uses high-throughput sequencing to provide a quantitative measurement of their regulatory functions at near-nucleotide resolution. We implemented RESA to identify sequence elements modulating mRNA stability during zebrafish embryogenesis...
December 26, 2016: Nature Methods
https://www.readbyqxmd.com/read/27992409/in-vivo-high-throughput-profiling-of-crispr-cpf1-activity
#9
Hui K Kim, Myungjae Song, Jinu Lee, A Vipin Menon, Soobin Jung, Young-Mook Kang, Jae W Choi, Euijeon Woo, Hyun C Koh, Jin-Wu Nam, Hyongbum Kim
CRISPR from Prevotella and Francisella 1 (Cpf1) is an effector endonuclease of the class 2 CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) gene editing system. We developed a method for evaluating Cpf1 activity, based on target sequence composition in mammalian cells, in a high-throughput manner. A library of >11,000 target sequence and guide RNA pairs was delivered into human cells using lentiviral vectors. Subsequent delivery of Cpf1 into this cell library induced insertions and deletions (indels) at the integrated synthetic target sequences, which allowed en masse evaluation of Cpf1 activity by using deep sequencing...
December 19, 2016: Nature Methods
https://www.readbyqxmd.com/read/27992408/cgal-a-temperature-robust-gal4-uas-system-for-caenorhabditis-elegans
#10
Han Wang, Jonathan Liu, Shahla Gharib, Cynthia M Chai, Erich M Schwarz, Navin Pokala, Paul W Sternberg
The GAL4-UAS system is a powerful tool for manipulating gene expression, but its application in Caenorhabditis elegans has not been described. Here we systematically optimize the system's three main components to develop a temperature-optimized GAL4-UAS system (cGAL) that robustly controls gene expression in C. elegans from 15 to 25 °C. We demonstrate this system's utility in transcriptional reporter analysis, site-of-action experiments and exogenous transgene expression; and we provide a basic driver and effector toolkit...
December 19, 2016: Nature Methods
https://www.readbyqxmd.com/read/27941785/nontargeted-in-vitro-metabolomics-for-high-throughput-identification-of-novel-enzymes-in-escherichia-coli
#11
Daniel C Sévin, Tobias Fuhrer, Nicola Zamboni, Uwe Sauer
Our understanding of metabolism is limited by a lack of knowledge about the functions of many enzymes. Here, we develop a high-throughput mass spectrometry approach to comprehensively profile proteins for in vitro enzymatic activity. Overexpressed or purified proteins are incubated in a supplemented metabolome extract containing hundreds of biologically relevant candidate substrates, and accumulating and depleting metabolites are determined by nontargeted mass spectrometry. By combining chemometrics and database approaches, we established an automated pipeline for unbiased annotation of the functions of novel enzymes...
December 12, 2016: Nature Methods
https://www.readbyqxmd.com/read/27941784/control-of-cerebral-ischemia-with-magnetic-nanoparticles
#12
Jie-Min Jia, Praveen D Chowdary, Xiaofei Gao, Bo Ci, Wenjun Li, Aditi Mulgaonkar, Erik J Plautz, Gedaa Hassan, Amit Kumar, Ann M Stowe, Shao-Hua Yang, Wei Zhou, Xiankai Sun, Bianxiao Cui, Woo-Ping Ge
The precise manipulation of microcirculation in mice can facilitate mechanistic studies of brain injury and repair after ischemia, but this manipulation remains a technical challenge, particularly in conscious mice. We developed a technology that uses micromagnets to induce aggregation of magnetic nanoparticles to reversibly occlude blood flow in microvessels. This allowed induction of ischemia in a specific cortical region of conscious mice of any postnatal age, including perinatal and neonatal stages, with precise spatiotemporal control but without surgical intervention of the skull or artery...
December 12, 2016: Nature Methods
https://www.readbyqxmd.com/read/27941783/simulation-based-comprehensive-benchmarking-of-rna-seq-aligners
#13
Giacomo Baruzzo, Katharina E Hayer, Eun Ji Kim, Barbara Di Camillo, Garret A FitzGerald, Gregory R Grant
Alignment is the first step in most RNA-seq analysis pipelines, and the accuracy of downstream analyses depends heavily on it. Unlike most steps in the pipeline, alignment is particularly amenable to benchmarking with simulated data. We performed a comprehensive benchmarking of 14 common splice-aware aligners for base, read, and exon junction-level accuracy and compared default with optimized parameters. We found that performance varied by genome complexity, and accuracy and popularity were poorly correlated...
December 12, 2016: Nature Methods
https://www.readbyqxmd.com/read/27918541/multidomain-structure-and-correlated-dynamics-determined-by-self-consistent-fret-networks
#14
Björn Hellenkamp, Philipp Wortmann, Florian Kandzia, Martin Zacharias, Thorsten Hugel
We present an approach that enables us to simultaneously access structure and dynamics of a multidomain protein in solution. Dynamic domain arrangements are experimentally determined by combining self-consistent networks of distance distributions with known domain structures. Local structural dynamics are correlated with the global arrangements by analyzing networks of time-resolved single-molecule fluorescence parameters. The strength of this hybrid approach is shown by an application to the flexible multidomain protein Hsp90...
December 5, 2016: Nature Methods
https://www.readbyqxmd.com/read/27918540/in-vivo-quantification-of-spatially-varying-mechanical-properties-in-developing-tissues
#15
Friedhelm Serwane, Alessandro Mongera, Payam Rowghanian, David A Kealhofer, Adam A Lucio, Zachary M Hockenbery, Otger Campàs
The mechanical properties of the cellular microenvironment and their spatiotemporal variations are thought to play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has yet been performed. Here we introduce a technique that employs biocompatible, magnetically responsive ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo...
December 5, 2016: Nature Methods
https://www.readbyqxmd.com/read/27918539/rapidly-evolving-homing-crispr-barcodes
#16
Reza Kalhor, Prashant Mali, George M Church
We present an approach for engineering evolving DNA barcodes in living cells. A homing guide RNA (hgRNA) scaffold directs the Cas9-hgRNA complex to the DNA locus of the hgRNA itself. We show that this homing CRISPR-Cas9 system acts as an expressed genetic barcode that diversifies its sequence and that the rate of diversification can be controlled in cultured cells. We further evaluate these barcodes in cell populations and show that they can be used to record lineage history and that the barcode RNA can be amplified in situ, a prerequisite for in situ sequencing...
December 5, 2016: Nature Methods
https://www.readbyqxmd.com/read/27798612/fast-volumetric-calcium-imaging-across-multiple-cortical-layers-using-sculpted-light
#17
Robert Prevedel, Aart J Verhoef, Alejandro J Pernía-Andrade, Siegfried Weisenburger, Ben S Huang, Tobias Nöbauer, Alma Fernández, Jeroen E Delcour, Peyman Golshani, Andrius Baltuska, Alipasha Vaziri
Although whole-organism calcium imaging in small and semi-transparent animals has been demonstrated, capturing the functional dynamics of large-scale neuronal circuits in awake behaving mammals at high speed and resolution has remained one of the main frontiers in systems neuroscience. Here we present a method based on light sculpting that enables unbiased single- and dual-plane high-speed (up to 160 Hz) calcium imaging as well as in vivo volumetric calcium imaging of a mouse cortical column (0.5 mm × 0.5 mm × 0...
October 31, 2016: Nature Methods
https://www.readbyqxmd.com/read/27798609/simultaneous-dual-color-fluorescence-lifetime-imaging-with-novel-red-shifted-fluorescent-proteins
#18
Tal Laviv, Benjamin B Kim, Jun Chu, Amy J Lam, Michael Z Lin, Ryohei Yasuda
We describe a red-shifted fluorescence resonance energy transfer (FRET) pair optimized for dual-color fluorescence lifetime imaging (FLIM). This pair utilizes a newly developed FRET donor, monomeric cyan-excitable red fluorescent protein (mCyRFP1), which has a large Stokes shift and a monoexponential fluorescence lifetime decay. When used together with EGFP-based biosensors, the new pair enables simultaneous imaging of the activities of two signaling molecules in single dendritic spines undergoing structural plasticity...
October 31, 2016: Nature Methods
https://www.readbyqxmd.com/read/27892959/novobreak-local-assembly-for-breakpoint-detection-in-cancer-genomes
#19
Zechen Chong, Jue Ruan, Min Gao, Wanding Zhou, Tenghui Chen, Xian Fan, Li Ding, Anna Y Lee, Paul Boutros, Junjie Chen, Ken Chen
We present novoBreak, a genome-wide local assembly algorithm that discovers somatic and germline structural variation breakpoints in whole-genome sequencing data. novoBreak consistently outperformed existing algorithms on real cancer genome data and on synthetic tumors in the ICGC-TCGA DREAM 8.5 Somatic Mutation Calling Challenge primarily because it more effectively utilized reads spanning breakpoints. novoBreak also demonstrated great sensitivity in identifying short insertions and deletions.
January 2017: Nature Methods
https://www.readbyqxmd.com/read/27892958/a-scored-human-protein-protein-interaction-network-to-catalyze-genomic-interpretation
#20
Taibo Li, Rasmus Wernersson, Rasmus B Hansen, Heiko Horn, Johnathan Mercer, Greg Slodkowicz, Christopher T Workman, Olga Rigina, Kristoffer Rapacki, Hans H Stærfeldt, Søren Brunak, Thomas S Jensen, Kasper Lage
Genome-scale human protein-protein interaction networks are critical to understanding cell biology and interpreting genomic data, but challenging to produce experimentally. Through data integration and quality control, we provide a scored human protein-protein interaction network (InWeb_InBioMap, or InWeb_IM) with severalfold more interactions (>500,000) and better functional biological relevance than comparable resources. We illustrate that InWeb_InBioMap enables functional interpretation of >4,700 cancer genomes and genes involved in autism...
January 2017: Nature Methods
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