Nature Protocols Contents: Volume 13 Number 12

TABLE OF CONTENTS December 2018 Volume 13, Issue 12 Editorial Review Articles Protocols Editorial Introducing our Tutorials    p2741 doi:10.1038/s41596-018-0078-6 Review Articles Tutorial: guidelines for the experimental design of single-cell RNA sequencing studies    pp2742 - 2757 Atefeh Lafzi, Catia Moutinho, Simone Picelli & Holger Heyn doi:10.1038/s41596-018-0073-y In this tutorial, the authors provide a comprehensive description of the considerations for designing single-cell transcriptomics studies, from sample preparation and single-cell RNA sequencing methodologies through data processing and analysis. Advertisement Searching for a new career? At Nature Research we frequently recruit talented individuals to join our editorial and publishing teams. If you have a PhD and a passion for science this may be the perfect career for you. Visit our research editorial and publishing careers website to discover more about what we do.   Protocols In vitro conversion of adult murine endothelial cells to hematopoietic stem cells    pp2758 - 2780 José Gabriel Barcia Durán, Raphaël Lis, Tyler M. Lu & Shahin Rafii doi:10.1038/s41596-018-0060-3 Adult endothelial cells from mice are reprogrammed to hematopoietic stem cells without transitioning through a pluripotent state. The protocol also describes in vitro and in vivo assays to confirm that the resulting cells function as expected. A computational framework to integrate high-throughput '-omics' datasets for the identification of potential mechanistic links    pp2781 - 2800 Helle Krogh Pedersen, Sofia K. Forslund, Valborg Gudmundsdottir, Anders Østergaard Petersen, Falk Hildebrand et al. doi:10.1038/s41596-018-0064-z This computational protocol offers a framework to integrate high-dimensional -omics datasets. A three-pronged association study integrating intestinal microbiome and serum metabolome data with measures of human host physiology is used as an example. Mitigating head motion artifact in functional connectivity MRI    pp2801 - 2826 Rastko Ciric, Adon F. G. Rosen, Guray Erus, Matthew Cieslak, Azeez Adebimpe et al. doi:10.1038/s41596-018-0065-y Ciric et al. describe a protocol for the removal of motion artifacts from functional MRI data. They introduce a software package that implements common denoising protocols and provides tools for assessing the efficacy of denoising. Blood–brain-barrier organoids for investigating the permeability of CNS therapeutics    pp2827 - 2843 Sonja Bergmann, Sean E. Lawler, Yuan Qu, Colin M. Fadzen, Justin M. Wolfe et al. doi:10.1038/s41596-018-0066-x Endothelial cells, pericytes and astrocytes are cocultured to generate organoids that reproduce many features of the blood–brain barrier. This protocol also describes how to analyze drug penetration into the organoids. CRISPR–Cas9-mediated genome editing in apple and grapevine    pp2844 - 2863 Yuriko Osakabe, Zhenchang Liang, Chong Ren, Chikako Nishitani, Keishi Osakabe et al. doi:10.1038/s41596-018-0067-9 This protocol provides approaches for applying the CRISPR–Cas9 system for genome editing in apple and grapevine plants, using both plasmid-mediated delivery of components and direct delivery of CRISPR–Cas9 ribonucleoproteins. A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein–protein interactions    pp2864 - 2889 Claudio Iacobucci, Michael Götze, Christian H. Ihling, Christine Piotrowski, Christian Arlt et al. doi:10.1038/s41596-018-0068-8 Valuable structural information can be derived by chemical cross-linking of proteins followed by mass spectrometry of the products. Iacobucci et al. use MS-cleavable reagents, enrichment by strong-cation-exchange chromatography, and LC-MS/MS analysis. Fast and high-resolution mapping of elastic properties of biomolecules and polymers with bimodal AFM    pp2890 - 2907 Simone Benaglia, Victor G. Gisbert, Alma P. Perrino, Carlos A. Amo & Ricardo Garcia doi:10.1038/s41596-018-0070-1 This protocol describes how to generate high-resolution maps of the elastic properties of biomolecules and polymers using bimodal AFM. The procedure covers sample preparation, bimodal AFM setup and calibration, and data acquisition and processing. Application and optimization of CRISPR–Cas9-mediated genome engineering in axolotl (Ambystoma mexicanum)    pp2908 - 2943 Ji-Feng Fei, Wilson Pak-Kin Lou, Dunja Knapp, Prayag Murawala, Tobias Gerber et al. doi:10.1038/s41596-018-0071-0 This protocol describes strategies for targeted genome modification in axolotls. Eggs are injected with a CAS9–gRNA ribonucleoprotein complex, which allows for efficient generation of knockout and knock-in animals and immediate phenotypic analysis. Speed breeding in growth chambers and glasshouses for crop breeding and model plant research    pp2944 - 2963 Sreya Ghosh, Amy Watson, Oscar E. Gonzalez-Navarro, Ricardo H. Ramirez-Gonzalez, Luis Yanes et al. doi:10.1038/s41596-018-0072-z This protocol describes procedures for speed-breeding approaches using growth cabinets and LED-supplemented glasshouses. The approaches can be used to accelerate crop research and are compatible with a wide variety of crops. Efficient and robust proteome-wide approaches for cross-linking mass spectrometry    pp2964 - 2990 Oleg Klykov, Barbara Steigenberger, Sibel Pektaş, Domenico Fasci, Albert J. R. Heck et al. doi:10.1038/s41596-018-0074-x Cross-linking of amino acids in close proximity on protein surfaces, followed by mass spectrometry, provides useful structural information. This protocol for using XlinkX can be applied for analysis of cultured cell samples or purified complexes. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins    pp2991 - 3017 Jonathan Elegheert, Ester Behiels, Benjamin Bishop, Suzanne Scott, Rachel E. Woolley et al. doi:10.1038/s41596-018-0075-9 This protocol describes a suite of lentiviral transfer plasmids that can be used for high-yield, time- and cost-efficient, and constitutive or inducible production of soluble and membrane proteins in mammalian cell lines. Use of human induced pluripotent stem cell–derived cardiomyocytes to assess drug cardiotoxicity    pp3018 - 3041 Arun Sharma, Wesley L. McKeithan, Ricardo Serrano, Tomoya Kitani, Paul W. Burridge et al. doi:10.1038/s41596-018-0076-8 This protocol describes the differentiation of hiPSCs into cardiomyocytes and subsequent cytotoxicity and contractility assays needed to calculate the 'cardiac safety index', which models the likelihood that a drug is cardiotoxic. Modular approach for resolving and mapping complex neural and other cellular structures and their associated deformation fields in three dimensions    pp3042 - 3064 Mark T. Scimone, Harry C. Cramer III, Eyal Bar-Kochba, Rodolfo Amezcua, Jonathan B. Estrada et al. doi:10.1038/s41596-018-0077-7 3D image segmentation and strain mapping are applied to topologically complex structures. As an example, we present the resolution of 3D strains on a culture containing neurons, astrocytes, and neural progenitors undergoing an in vitro injury event. Advertisement Nature Briefing is an essential round-up of science news, opinion and analysis, free in your inbox every weekday. 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