Nature Methods Contents: December 2016 Volume 13 pp 959 - 1055

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Advertisement ZEISS LSM 880 with Airyscan - Your new standard for fast and gentle confocal imaging Learn how to improve signal-to-noise ratio, resolution and speed of your confocal imaging. Discover how the Fast mode for Airyscan compares to traditional confocal detection and download our free white paper with more information TABLE OF CONTENTS December 2016 Volume 13, Issue 12 In This Issue Editorial This Month Correspondence Research Highlights Technology Feature News and Views Brief Communications Articles Erratum Application Note Advertisement Innova® Shakers: Versatile, dependable, and easy to use For over 60 years, New Brunswick Shakers have been synonymous with quality construction and durability. The dependable operation is due in large part to the Eppendorf triple-eccentric counterbalanced drive; drive shafts are machined to tolerances of 5 micrometers, ensuring stable and vibration-free operation, often over decades of continuous use. Subscribe   Facebook   RSS   Recommend to library   Twitter   Advertisement Maximize culture quality and minimize daily feeding.  StemBeads® are developed and tested by scientists who understand the complexity of cells and the importance of high quality reagents. Reduce Spontaneous Differentiation and Cell Death Improve Culture Quality Use your Favorite Media Media savings of up to 67% Save Time and Money Watch our video and learn more    Advertisement Pre-made CRISPR-Cas9 Stable Cell Lines Stably expressing CRISPR Cas9. Ideal for CRISPR sgRNA validation, library screening, and sgRNA mediated knockouts and knockins Human: H1299 - Cas9, HEK293T - Cas9, HeLa - Cas9, A549 - Cas9, MCF-7 - Cas9, MDA-MB-231 - Cas9, HepG2 - Cas9, AGS - Cas9, BXPC-3 - Cas9 Mouse: Neuro2a - Cas9  Learn more >>   In This Issue Top In This Issue    Editorial Top Meta-analysis in basic biology   p959 doi:10.1038/nmeth.4102 Meta-analysis is common in clinical research, less so in basic biology, but it is also proving useful in some basic research contexts. It should help improve research reproducibility. This Month Top The Author File: Alipasha Vaziri   p961 Vivien Marx doi:10.1038/nmeth.4085 A physicist handy with charcoal and ink uses sculpted light to image the brain. Correspondence Top Temporal accumulation analysis provides simplified artifact-free analysis of membrane-protein nanoclusters   pp963 - 964 Christoph Spahn, Frank Herrmannsdorfer, Thomas Kuner and Mike Heilemann doi:10.1038/nmeth.4065 moFF: a robust and automated approach to extract peptide ion intensities   pp964 - 966 Andrea Argentini, Ludger J E Goeminne, Kenneth Verheggen, Niels Hulstaert, An Staes et al. doi:10.1038/nmeth.4075 OmniPath: guidelines and gateway for literature-curated signaling pathway resources   pp966 - 967 Dénes Türei, Tamás Korcsmáros and Julio Saez-Rodriguez doi:10.1038/nmeth.4077 Research Highlights Top A blood less primitive Cord blood provides important clues for directing human pluripotent stem cells to become pre-hematopoietic stem cells. Single-molecule force analysis, unplugged A nanoscopic force clamp enables high-throughput single-molecule analysis of DNA under tension without connection to a macroscopic instrument. REXER helps design bacterial genomes A CRISPR-Cas9-based approach identifies synonymous codons that can be reassigned to encode new chemical building blocks. Proteome quantification compared Careful benchmarking improves software methods for analyzing data-independent-acquisition mass spectrometry data. A pluripotent approach A combination of protein and DNA isolation methods expands our grasp of cellular reprogramming. Extending super-resolution's FOV An optimized illumination strategy allows for uniform single-molecule localization microscopy over large regions. Methods in Brief Hi-res spatial gene expression in the brain | Man-made mouse eggs | Laser ablation for growing protein crystals | Annotating RNAs by the company they keep Tools in Brief CRISPR screens tackle the noncoding genome | Instant tracking of single-molecule position and orientation | Three steps to installing authentic protein modifications | Nanokits for single cells Methods JOBS of the week Post-doctoral fellow with a PhD in biological sciences or bioinformatics (m / f) Max Planck Institute of Psychiatry Postdoc Position in Bioinformatics Ludwig Boltzmann Institute Applied Diagnostics Positions at Center for Precision Medicine, The First Affiliated Hospital of Wenzhou Medical University The First Affiliated Hospital of Wenzhou Medical University Senior University Lecturer in Geographic Information Science specialising in Geomatics Lund University University Lectureship in Multi-Scale, Multi-Dimensional Imaging of Natural and Synthetic Materials University of Cambridge More Science jobs from Technology Feature Top Microscopy: OpenSPIM 2.0   pp979 - 982 Vivien Marx doi:10.1038/nmeth.4070 A maturing open hardware and open-source software movement seeks to expand DIY light-sheet microscopy. News and Views Top CRISPR-Cas9-AID base editor is a powerful gain-of-function screening tool   pp983 - 984 Cem Kuscu and Mazhar Adli doi:10.1038/nmeth.4076 Combining CRISPR-Cas9 with an enzyme that induces somatic hypermutations allows the rapid generation of diverse variants for gain-of-function screens. See also: Article by Ma et al. | Article by Hess et al. Brief Communications Top Bright photoactivatable fluorophores for single-molecule imaging   pp985 - 988 Jonathan B Grimm, Brian P English, Heejun Choi, Anand K Muthusamy, Brian P Mehl et al. doi:10.1038/nmeth.4034 Photoactivatable derivatives of the bright and photostable Janelia Fluor dyes enable improved multicolor single-particle tracking and facile localization microscopy in cells. Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins   pp989 - 992 Tal Laviv, Benjamin B Kim, Jun Chu, Amy J Lam, Michael Z Lin et al. doi:10.1038/nmeth.4046 Two red fluorescent proteins with long Stokes shift enable simultaneous multicolor 2p imaging. CyRFP1 is well-suited for 2p structural imaging, and FRET sensors made with mCyRFP1 and mMaroon1enable multicolor 2pFLIM in brain slices. Also online, a paper by Bajar et al. reports the development of mMaroon1. See also: Brief Communication by Bajar et al. Fluorescent indicators for simultaneous reporting of all four cell cycle phases   pp993 - 996 Bryce T Bajar, Amy J Lam, Ryan K Badiee, Young-Hee Oh, Jun Chu et al. doi:10.1038/nmeth.4045 The far-red fluorescent protein mMaroon1 and a reporter based on stem-loop binding protein enables the generation of Fucci4, a 4-color cell cycle reporter system that can be used to distinguish all phases of the cell cycle. Also online, a paper by Laviv et al. uses mMaroon1 as a FRET acceptor for the newly developed CyRFP1. See also: Brief Communication by Laviv et al. Genetic code expansion for multiprotein complex engineering   pp997 - 1000 Christine Koehler, Paul F Sauter, Mirella Wawryszyn, Gemma Estrada Girona, Kapil Gupta et al. doi:10.1038/nmeth.4032 A method for producing multiprotein complexes engineered with site-specifically introduced noncanonical amino acids is described, enabling applications in biochemical and biophysical analysis, as well as in biotechnology. Random-access scanning microscopy for 3D imaging in awake behaving animals   pp1001 - 1004 K M Naga Srinivas Nadella, Hana Roš, Chiara Baragli, Victoria A Griffiths, George Konstantinou et al. doi:10.1038/nmeth.4033 Random-access line scanning enables neural activity to be monitored at high speed in neurons and dendrites that are sparsely distributed in three dimensions. The approach is demonstrated in behaving mice. Comparison of high-throughput sequencing data compression tools   pp1005 - 1008 Ibrahim Numanagić, James K Bonfield, Faraz Hach, Jan Voges, Jörn Ostermann et al. doi:10.1038/nmeth.4037 A team of international scientists benchmark current compression methods for high-throughput sequencing data. Micro-C XL: assaying chromosome conformation from the nucleosome to the entire genome   pp1009 - 1011 Tsung-Han S Hsieh, Geoffrey Fudenberg, Anton Goloborodko and Oliver J Rando doi:10.1038/nmeth.4025 The combination of short and long crosslinkers during chromosome conformation capture allows the interrogation of structure from the nucleosome to the chromosome-wide level in yeast. Advertisement Nature Index 2016 Australia & New Zealand Nature Index 2016 Australia and New Zealand highlights the quality natural science research within these countries. This supplement examines the cities and institutions that largely contribute to the research as well as the factors that drive their success.  Access the free supplement for six months!     Articles Top ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing   pp1013 - 1020 Xingqi Chen, Ying Shen, Will Draper, Jason D Buenrostro, Ulrike Litzenburger et al. doi:10.1038/nmeth.4031 The covalent insertion of fluorophore-labeled DNA adaptors by Tn5 transposase into open chromatin allows its imaging and subsequent analysis by sequencing from exactly the same samples. Fast volumetric calcium imaging across multiple cortical layers using sculpted light   pp1021 - 1028 Robert Prevedel, Aart J Verhoef, Alejandro J Pernía-Andrade, Siegfried Weisenburger, Ben S Huang et al. doi:10.1038/nmeth.4040 Two-photon scanning microscopy is inherently slow and thus limits volumetric calcium imaging. Prevedel et al. achieve increased volumetric imaging speed by tailoring the excitation volume via light sculpting. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells   pp1029 - 1035 Yunqing Ma, Jiayuan Zhang, Weijie Yin, Zhenchao Zhang, Yan Song et al. doi:10.1038/nmeth.4027 Fusing a cytidine deaminase to dCas9 allows for a forward genetic screen in mammalian cells that identifies mutations conferring drug resistance. See also: News and Views by Kuscu & Adli Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells   pp1036 - 1042 Gaelen T Hess, Laure Frésard, Kyuho Han, Cameron H Lee, Amy Li et al. doi:10.1038/nmeth.4038 Recruiting a hyperactive cytidine deaminase via the guide RNA to dCas9 allows for the introduction of diverse point mutations at the CRISPR target locus to create complex libraries of variants for protein engineering or dissection of protein function. See also: News and Views by Kuscu & Adli Complex transcriptional modulation with orthogonal and inducible dCas9 regulators   pp1043 - 1049 Yuchen Gao, Xin Xiong, Spencer Wong, Emeric J Charles, Wendell A Lim et al. doi:10.1038/nmeth.4042 The combination of orthogonal dCas9 with two chemical-inducible dimerization systems allows precise induction of gene activation and repression as well as the creation of Boolean logic gates. Phased diploid genome assembly with single-molecule real-time sequencing   pp1050 - 1054 Chen-Shan Chin, Paul Peluso, Fritz J Sedlazeck, Maria Nattestad, Gregory T Concepcion et al. doi:10.1038/nmeth.4035 The open-source FALCON and FALCON-Unzip software utilize long-read sequencing data to generate contiguous, accurate and phased diploid assemblies, even from genomes that are highly heterozygous. 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