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Nature Methods

Matthias Meurer, Yuanqiang Duan, Ehud Sass, Ilia Kats, Konrad Herbst, Benjamin C Buchmuller, Verena Dederer, Florian Huber, Daniel Kirrmaier, Martin Štefl, Koen Van Laer, Tobias P Dick, Marius K Lemberg, Anton Khmelinskii, Emmanuel D Levy, Michael Knop
Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.
July 9, 2018: Nature Methods
Francesca Pennacchietti, Ekaterina O Serebrovskaya, Aline R Faro, Irina I Shemyakina, Nina G Bozhanova, Alexey A Kotlobay, Nadya G Gurskaya, Andreas Bodén, Jes Dreier, Dmitry M Chudakov, Konstantin A Lukyanov, Vladislav V Verkhusha, Alexander S Mishin, Ilaria Testa
Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells.
July 9, 2018: Nature Methods
Uri Weill, Ido Yofe, Ehud Sass, Bram Stynen, Dan Davidi, Janani Natarajan, Reut Ben-Menachem, Zohar Avihou, Omer Goldman, Nofar Harpaz, Silvia Chuartzman, Kiril Kniazev, Barbara Knoblach, Janina Laborenz, Felix Boos, Jacqueline Kowarzyk, Shifra Ben-Dor, Einat Zalckvar, Johannes M Herrmann, Richard A Rachubinski, Ophry Pines, Doron Rapaport, Stephen W Michnick, Emmanuel D Levy, Maya Schuldiner
Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore...
July 9, 2018: Nature Methods
Mo Huang, Jingshu Wang, Eduardo Torre, Hannah Dueck, Sydney Shaffer, Roberto Bonasio, John I Murray, Arjun Raj, Mingyao Li, Nancy R Zhang
In single-cell RNA sequencing (scRNA-seq) studies, only a small fraction of the transcripts present in each cell are sequenced. This leads to unreliable quantification of genes with low or moderate expression, which hinders downstream analysis. To address this challenge, we developed SAVER (single-cell analysis via expression recovery), an expression recovery method for unique molecule index (UMI)-based scRNA-seq data that borrows information across genes and cells to provide accurate expression estimates for all genes...
June 25, 2018: Nature Methods
Antonio Lentini, Cathrine Lagerwall, Svante Vikingsson, Heidi K Mjoseng, Karolos Douvlataniotis, Hartmut Vogt, Henrik Green, Richard R Meehan, Mikael Benson, Colm E Nestor
DNA immunoprecipitation followed by sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, the results of independent DIP-seq studies often show considerable variation between profiles of the same genome and between profiles obtained by alternative methods. Here we show that these differences are primarily due to the intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50-99% of regions identified as 'enriched' for DNA modifications in DIP-seq data...
June 25, 2018: Nature Methods
Amaro Taylor-Weiner, Chip Stewart, Thomas Giordano, Mendy Miller, Mara Rosenberg, Alyssa Macbeth, Niall Lennon, Esther Rheinbay, Dan-Avi Landau, Catherine J Wu, Gad Getz
Comparison of sequencing data from a tumor sample with data from a matched germline control is a key step for accurate detection of somatic mutations. Detection sensitivity for somatic variants is greatly reduced when the matched normal sample is contaminated with tumor cells. To overcome this limitation, we developed deTiN, a method that estimates the tumor-in-normal (TiN) contamination level and, in cases affected by contamination, improves sensitivity by reclassifying initially discarded variants as somatic...
June 25, 2018: Nature Methods
Pei-Hsun Wu, Dikla Raz-Ben Aroush, Atef Asnacios, Wei-Chiang Chen, Maxim E Dokukin, Bryant L Doss, Pauline Durand-Smet, Andrew Ekpenyong, Jochen Guck, Nataliia V Guz, Paul A Janmey, Jerry S H Lee, Nicole M Moore, Albrecht Ott, Yeh-Chuin Poh, Robert Ros, Mathias Sander, Igor Sokolov, Jack R Staunton, Ning Wang, Graeme Whyte, Denis Wirtz
The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching...
June 18, 2018: Nature Methods
Taibo Li, April Kim, Joseph Rosenbluh, Heiko Horn, Liraz Greenfeld, David An, Andrew Zimmer, Arthur Liberzon, Jon Bistline, Ted Natoli, Yang Li, Aviad Tsherniak, Rajiv Narayan, Aravind Subramanian, Ted Liefeld, Bang Wong, Dawn Thompson, Sarah Calvo, Steve Carr, Jesse Boehm, Jake Jaffe, Jill Mesirov, Nir Hacohen, Aviv Regev, Kasper Lage
Functional genomics networks are widely used to identify unexpected pathway relationships in large genomic datasets. However, it is challenging to compare the signal-to-noise ratios of different networks and to identify the optimal network with which to interpret a particular genetic dataset. We present GeNets, a platform in which users can train a machine-learning model (Quack) to carry out these comparisons and execute, store, and share analyses of genetic and RNA-sequencing datasets.
June 18, 2018: Nature Methods
Sebastian Virreira Winter, Florian Meier, Christoph Wichmann, Juergen Cox, Matthias Mann, Felix Meissner
We developed EASI-tag (easily abstractable sulfoxide-based isobaric-tag), a new type of amine-derivatizing and sulfoxide-containing isobaric labeling reagents for highly accurate quantitative proteomics analysis using mass spectrometry. We observed that EASI-tag labels dissociate at low collision energy and generate peptide-coupled, interference-free reporter ions with high yield. Efficient isolation of 12 C precursors and quantification at the MS2 level allowed accurate determination of quantitative differences between up to six multiplexed samples...
June 18, 2018: Nature Methods
Jungkap Park, Paul D Piehowski, Christopher Wilkins, Mowei Zhou, Joshua Mendoza, Grant M Fujimoto, Bryson C Gibbons, Jared B Shaw, Yufeng Shen, Anil K Shukla, Ronald J Moore, Tao Liu, Vladislav A Petyuk, Nikola Tolić, Ljiljana Paša-Tolić, Richard D Smith, Samuel H Payne, Sangtae Kim
In the version of this article initially published, the authors erroneously reported the search mode that was used for ProSightPC 3.0 in the Online Methods and in Supplementary Table 3.
June 13, 2018: Nature Methods
Jean-Charles Boisset, Judith Vivié, Dominic Grün, Mauro J Muraro, Anna Lyubimova, Alexander van Oudenaarden
A cell's function is influenced by the environment, or niche, in which it resides. Studies of niches usually require assumptions about the cell types present, which impedes the discovery of new cell types or interactions. Here we describe ProximID, an approach for building a cellular network based on physical cell interaction and single-cell mRNA sequencing, and show that it can be used to discover new preferential cellular interactions without prior knowledge of component cell types. ProximID found specific interactions between megakaryocytes and mature neutrophils and between plasma cells and myeloblasts and/or promyelocytes (precursors of neutrophils) in mouse bone marrow, and it identified a Tac1+ enteroendocrine cell-Lgr5+ stem cell interaction in small intestine crypts...
May 21, 2018: Nature Methods
Shane R Ellis, Martin R L Paine, Gert B Eijkel, Josch K Pauling, Peter Husen, Mark W Jervelund, Martin Hermansson, Christer S Ejsing, Ron M A Heeren
We report a method that enables automated data-dependent acquisition of lipid tandem mass spectrometry data in parallel with a high-resolution mass spectrometry imaging experiment. The method does not increase the total image acquisition time and is combined with automatic structural assignments. This lipidome-per-pixel approach automatically identified and validated 104 unique molecular lipids and their spatial locations from rat cerebellar tissue.
May 21, 2018: Nature Methods
Keith R Anderson, Maximilian Haeussler, Colin Watanabe, Vasantharajan Janakiraman, Jessica Lund, Zora Modrusan, Jeremy Stinson, Qixin Bei, Andrew Buechler, Charles Yu, Sobha R Thamminana, Lucinda Tam, Michael-Anne Sowick, Tuija Alcantar, Natasha O'Neil, Jinjie Li, Linda Ta, Lisa Lima, Merone Roose-Girma, Xin Rairdan, Steffen Durinck, Søren Warming
Despite widespread use of CRISPR, comprehensive data on the frequency and impact of Cas9-mediated off-targets in modified rodents are limited. Here we present deep-sequencing data from 81 genome-editing projects on mouse and rat genomes at 1,423 predicted off-target sites, 32 of which were confirmed, and show that high-fidelity Cas9 versions reduced off-target mutation rates in vivo. Using whole-genome sequencing data from ten mouse embryos, treated with a single guide RNA (sgRNA), and from their genetic parents, we found 43 off-targets, 30 of which were predicted by an adapted version of GUIDE-seq...
May 21, 2018: Nature Methods
Min Guo, Panagiotis Chandris, John Paul Giannini, Adam J Trexler, Robert Fischer, Jiji Chen, Harshad D Vishwasrao, Ivan Rey-Suarez, Yicong Wu, Xufeng Wu, Clare M Waterman, George H Patterson, Arpita Upadhyaya, Justin W Taraska, Hari Shroff
We combined instant structured illumination microscopy (iSIM) with total internal reflection fluorescence microscopy (TIRFM) in an approach referred to as instant TIRF-SIM, thereby improving the lateral spatial resolution of TIRFM to 115 ± 13 nm without compromising speed, and enabling imaging frame rates up to 100 Hz over hundreds of time points. We applied instant TIRF-SIM to multiple live samples and achieved rapid, high-contrast super-resolution imaging close to the coverslip surface.
May 7, 2018: Nature Methods
Florian Meier, Philipp E Geyer, Sebastian Virreira Winter, Juergen Cox, Matthias Mann
Great advances have been made in sensitivity and acquisition speed on the Orbitrap mass analyzer, enabling increasingly deep proteome coverage. However, these advances have been mainly limited to the MS2 level, whereas ion beam sampling for the MS1 scans remains extremely inefficient. Here we report a data-acquisition method, termed BoxCar, in which filling multiple narrow mass-to-charge segments increases the mean ion injection time more than tenfold as compared to that of a standard full scan. In 1-h analyses, the method provided MS1-level evidence for more than 90% of the proteome of a human cancer cell line that had previously been identified in 24 fractions, and it quantified more than 6,200 proteins in ten of ten replicates...
May 7, 2018: Nature Methods
Samuel A Myers, Jason Wright, Ryan Peckner, Brian T Kalish, Feng Zhang, Steven A Carr
Regulation of gene expression is primarily controlled by changes in the proteins that occupy genes' regulatory elements. We developed genomic locus proteomics (GLoPro), in which we combine CRISPR-based genome targeting, proximity labeling, and quantitative proteomics to discover proteins associated with a specific genomic locus in native cellular contexts.
May 7, 2018: Nature Methods
Xin D Gao, Li-Chun Tu, Aamir Mir, Tomás Rodriguez, Yuehe Ding, John Leszyk, Job Dekker, Scott A Shaffer, Lihua Julie Zhu, Scot A Wolfe, Erik J Sontheimer
Mapping proteomic composition at distinct genomic loci in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 biotinylation at genomic elements by restricted spatial tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. C-BERST enables the high-throughput identification of proteins associated with specific sequences, thereby facilitating annotation of these factors and their roles.
May 7, 2018: Nature Methods
Vivien Marx
No abstract text is available yet for this article.
July 2018: Nature Methods
Zachary J Lapin
No abstract text is available yet for this article.
July 2018: Nature Methods
Lei Tang
No abstract text is available yet for this article.
July 2018: Nature Methods
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