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Nature Protocols

Reazur Rahman, Weijin Xu, Hua Jin, Michael Rosbash
RNA-binding proteins (RBPs) accompany RNA from birth to death, affecting RNA biogenesis and functions. Identifying RBP-RNA interactions is essential to understanding their complex roles in different cellular processes. However, detecting in vivo RNA targets of RBPs, especially in a small number of discrete cells, has been a technically challenging task. We previously developed a novel technique called TRIBE (targets of RNA-binding proteins identified by editing) to overcome this problem. TRIBE expresses a fusion protein consisting of a queried RBP and the catalytic domain of the RNA-editing enzyme ADAR (adenosine deaminase acting on RNA) (ADARcd), which marks target RNA transcripts by converting adenosine to inosine near the RBP binding sites...
July 16, 2018: Nature Protocols
Tommi Huhtamäki, Xuelin Tian, Juuso T Korhonen, Robin H A Ras
Wetting, the process of water interacting with a surface, is critical in our everyday lives and in many biological and technological systems. The contact angle is the angle at the interface where water, air and solid meet, and its value is a measure of how likely the surface is to be wetted by the water. Low contact-angle values demonstrate a tendency of the water to spread and adhere to the surface, whereas high contact-angle values show the surface's tendency to repel water. The most common method for surface-wetting characterization is sessile-drop goniometry, due to its simplicity...
July 9, 2018: Nature Protocols
Philipp Mertins, Lauren C Tang, Karsten Krug, David J Clark, Marina A Gritsenko, Lijun Chen, Karl R Clauser, Therese R Clauss, Punit Shah, Michael A Gillette, Vladislav A Petyuk, Stefani N Thomas, D R Mani, Filip Mundt, Ronald J Moore, Yingwei Hu, Rui Zhao, Michael Schnaubelt, Hasmik Keshishian, Matthew E Monroe, Zhen Zhang, Namrata D Udeshi, Deepak Mani, Sherri R Davies, R Reid Townsend, Daniel W Chan, Richard D Smith, Hui Zhang, Tao Liu, Steven A Carr
Here we present an optimized workflow for global proteome and phosphoproteome analysis of tissues or cell lines that uses isobaric tags (TMT (tandem mass tags)-10) for multiplexed analysis and relative quantification, and provides 3× higher throughput than iTRAQ (isobaric tags for absolute and relative quantification)-4-based methods with high intra- and inter-laboratory reproducibility. The workflow was systematically characterized and benchmarked across three independent laboratories using two distinct breast cancer subtypes from patient-derived xenograft models to enable assessment of proteome and phosphoproteome depth and quantitative reproducibility...
July 9, 2018: Nature Protocols
Arjen Stolk, Sandon Griffin, Roemer van der Meij, Callum Dewar, Ignacio Saez, Jack J Lin, Giovanni Piantoni, Jan-Mathijs Schoffelen, Robert T Knight, Robert Oostenveld
Human intracranial electroencephalography (iEEG) recordings provide data with much greater spatiotemporal precision than is possible from data obtained using scalp EEG, magnetoencephalography (MEG), or functional MRI. Until recently, the fusion of anatomical data (MRI and computed tomography (CT) images) with electrophysiological data and their subsequent analysis have required the use of technologically and conceptually challenging combinations of software. Here, we describe a comprehensive protocol that enables complex raw human iEEG data to be converted into more readily comprehensible illustrative representations...
July 9, 2018: Nature Protocols
Sarah Ellys Harrison, Berna Sozen, Magdalena Zernicka-Goetz
Mammalian embryogenesis requires the coordination of embryonic and extra-embryonic tissues to enable implantation into the uterus and post-implantation development to establish the body plan. Mouse embryonic stem cells (ESCs) are a useful tool for studying pluripotent embryonic tissue in vitro. However, they cannot undertake correct embryogenesis alone. Many attempts to model the early embryo in vitro involve the aggregation of ESCs into spheroids of variable size and cell number that undertake germ-layer specification but fail to recapitulate the characteristic architecture and arrangement of tissues of the early embryo...
July 9, 2018: Nature Protocols
Jie Chao, Honglu Zhang, Yikang Xing, Qian Li, Huajie Liu, Lihua Wang, Lianhui Wang, Chunhai Fan
Atomic force microscopy (AFM)-based nanomechanical imaging provides a high-resolution approach for imaging biomolecules with nanometer resolution. Nevertheless, the lack of appropriate nanomechanical labels poses a limit to biological applications. Here, we describe how to generate a set of shape-resolved nanomechanical labels by exploiting self-assembled DNA origami technology. By designing 'mediator' strands that can hybridize with both the origami shapes and the target DNA, these origami shape IDs can be used to site-specifically label genomic DNA with high efficiency and high throughput...
July 9, 2018: Nature Protocols
Meng-Ying Liu, Chun-Yu Yin, Li-Juan Zhu, Xian-Hui Zhu, Chu Xu, Chun-Xia Luo, Hongshan Chen, Dong-Ya Zhu, Qi-Gang Zhou
Anhedonia is the inability to experience pleasure from rewarding or enjoyable activities and is a core symptom of depression in humans. Here, we describe a protocol for the measurement of anhedonia in mice, in which anhedonia is measured by a sucrose preference test (SPT) based on a two-bottle choice paradigm. A reduction in the sucrose preference ratio in experimental relative to control mice is indicative of anhedonia. To date, inconsistent and variable results have been reported following the use of the SPT by different groups, probably due to the use of different protocols and equipment...
July 9, 2018: Nature Protocols
Florian A Dehmelt, Adam von Daranyi, Claire Leyden, Aristides B Arrenberg
Reliable measurement of spontaneous and evoked eye movement is critical for behavioral vision research. Zebrafish are increasingly used as a model organism for visual neural circuits, but ready-to-use eye-tracking solutions are scarce. Here, we present a protocol for automated real-time measurement of angular horizontal eye position in up to six immobilized larval fish using a custom-built LabVIEW-based software, ZebEyeTrack. We provide its customizable source code, as well as a streamlined and compiled version, ZebEyeTrack Light...
July 9, 2018: Nature Protocols
Bing Jiang, Demin Duan, Lizeng Gao, Mengjie Zhou, Kelong Fan, Yan Tang, Juqun Xi, Yuhai Bi, Zhou Tong, George Fu Gao, Ni Xie, Aifa Tang, Guohui Nie, Minmin Liang, Xiyun Yan
Nanozymes are nanomaterials exhibiting intrinsic enzyme-like characteristics that have increasingly attracted attention, owing to their high catalytic activity, low cost and high stability. This combination of properties has enabled a broad spectrum of applications, ranging from biological detection assays to disease diagnosis and biomedicine development. Since the intrinsic peroxidase activity of Fe3 O4 nanoparticles (NPs) was first reported in 2007, >40 types of nanozymes have been reported that possess peroxidase-, oxidase-, haloperoxidase- or superoxide dismutase-like catalytic activities...
July 2, 2018: Nature Protocols
Zsuzsanna Orbán-Németh, Rebecca Beveridge, David M Hollenstein, Evelyn Rampler, Thomas Stranzl, Otto Hudecz, Johannes Doblmann, Peter Schlögelhofer, Karl Mechtler
No abstract text is available yet for this article.
June 25, 2018: Nature Protocols
Alexandre M J J Bonvin, Ezgi Karaca, Panagiotis L Kastritis, João P G L M Rodrigues
No abstract text is available yet for this article.
June 25, 2018: Nature Protocols
Zsuzsanna Orbán-Németh, Rebecca Beveridge, David M Hollenstein, Evelyn Rampler, Thomas Stranzl, Otto Hudecz, Johannes Doblmann, Peter Schlögelhofer, Karl Mechtler
In the version of this article initially published online, the authors used incorrectly defined restraints for specifying the distance between residues when using the HADDOCK portal. Following the publication of a Correspondence by the developers of the HADDOCK portal (Nat. Protoc., 2018) and a Reply by the authors of the Protocol (Nat. Protoc., 2018), the syntax in step 21 has been corrected. In addition, the input files (available as Supplementary Data 5-7) have been replaced...
June 25, 2018: Nature Protocols
Samira Musah, Nikolaos Dimitrakakis, Diogo M Camacho, George M Church, Donald E Ingber
Protocols have been established to direct the differentiation of human induced pluripotent stem (iPS) cells into nephron progenitor cells and organoids containing many types of kidney cells, but it has been difficult to direct the differentiation of iPS cells to form specific types of mature human kidney cells with high yield. Here, we describe a detailed protocol for the directed differentiation of human iPS cells into mature, post-mitotic kidney glomerular podocytes with high (>90%) efficiency within 26 d and under chemically defined conditions, without genetic manipulations or subpopulation selection...
July 2018: Nature Protocols
Cheng Lei, Hirofumi Kobayashi, Yi Wu, Ming Li, Akihiro Isozaki, Atsushi Yasumoto, Hideharu Mikami, Takuro Ito, Nao Nitta, Takeaki Sugimura, Makoto Yamada, Yutaka Yatomi, Dino Di Carlo, Yasuyuki Ozeki, Keisuke Goda
The ability to rapidly assay morphological and intracellular molecular variations within large heterogeneous populations of cells is essential for understanding and exploiting cellular heterogeneity. Optofluidic time-stretch microscopy is a powerful method for meeting this goal, as it enables high-throughput imaging flow cytometry for large-scale single-cell analysis of various cell types ranging from human blood to algae, enabling a unique class of biological, medical, pharmaceutical, and green energy applications...
July 2018: Nature Protocols
Christian Wiwie, Jan Baumbach, Richard Röttger
Clustering is a popular technique for discovering groups of similar objects in large datasets. It is nowadays applied in all areas of life sciences, from biomedicine to physics. However, designing high-quality cluster analyses is a tedious and complicated task with manifold choices along the way. As a cluster analysis is often the first step of a succeeding downstream analysis, the clustering must be reliable, reproducible, and of the highest quality. To address these challenges, we recently developed ClustEval, an integrated and extensible platform for the automated and standardized design and execution of complex cluster analyses...
June 2018: Nature Protocols
Yupeng Cun, Tsun-Po Yang, Viktor Achter, Ulrich Lang, Martin Peifer
The genomes of cancer cells constantly change during pathogenesis. This evolutionary process can lead to the emergence of drug-resistant mutations in subclonal populations, which can hinder therapeutic intervention in patients. Data derived from massively parallel sequencing can be used to infer these subclonal populations using tumor-specific point mutations. The accurate determination of copy-number changes and tumor impurity is necessary to reliably infer subclonal populations by mutational clustering. This protocol describes how to use Sclust, a copy-number analysis method with a recently developed mutational clustering approach...
June 2018: Nature Protocols
Julien Duez, Mario Carucci, Irene Garcia-Barbazan, Matias Corral, Oscar Perez, Jesus Luis Presa, Benoit Henry, Camille Roussel, Papa Alioune Ndour, Noemi Bahamontes Rosa, Laura Sanz, Francisco-Javier Gamo, Pierre Buffet
The mechanical retention of rigid erythrocytes in the spleen is central in major hematological diseases such as hereditary spherocytosis, sickle-cell disease and malaria. Here, we describe the use of microsphiltration (microsphere filtration) to assess erythrocyte deformability in hundreds to thousands of samples in parallel, by filtering them through microsphere layers in 384-well plates adapted for the discovery of compounds that stiffen Plasmodium falciparum gametocytes, with the aim of interrupting malaria transmission...
June 2018: Nature Protocols
Antonio Z Politi, Yin Cai, Nike Walther, M Julius Hossain, Birgit Koch, Malte Wachsmuth, Jan Ellenberg
The ability to tag a protein at its endogenous locus with a fluorescent protein (FP) enables quantitative understanding of protein dynamics at the physiological level. Genome-editing technology has now made this powerful approach routinely applicable to mammalian cells and many other model systems, thereby opening up the possibility to systematically and quantitatively map the cellular proteome in four dimensions. 3D time-lapse confocal microscopy (4D imaging) is an essential tool for investigating spatial and temporal protein dynamics; however, it lacks the required quantitative power to make the kind of absolute and comparable measurements required for systems analysis...
June 2018: Nature Protocols
Kunhong Xiao, Yang Zhao, Minjung Choi, Hongda Liu, Adi Blanc, Jiang Qian, Thomas J Cahill, Xue Li, Yunfang Xiao, Lisa J Clark, Sheng Li
Many cellular functions necessitate structural assemblies of two or more associated proteins. The structural characterization of protein complexes using standard methods, such as X-ray crystallography, is challenging. Herein, we describe an orthogonal approach using hydrogen-deuterium-exchange mass spectrometry (HDXMS), cross-linking mass spectrometry (CXMS), and disulfide trapping to map interactions within protein complexes. HDXMS measures changes in solvent accessibility and hydrogen bonding upon complex formation; a decrease in HDX rate could account for newly formed intermolecular or intramolecular interactions...
June 2018: Nature Protocols
Kassandra Kisler, Divna Lazic, Melanie D Sweeney, Shane Plunkett, Mirna El Khatib, Sergei A Vinogradov, David A Boas, Sava Sakadži, Berislav V Zlokovic
Cerebrovascular dysfunction has an important role in the pathogenesis of multiple brain disorders. Measurement of hemodynamic responses in vivo can be challenging, particularly as techniques are often not described in sufficient detail and vary between laboratories. We present a set of standardized in vivo protocols that describe high-resolution two-photon microscopy and intrinsic optical signal (IOS) imaging to evaluate capillary and arteriolar responses to a stimulus, regional hemodynamic responses, and oxygen delivery to the brain...
June 2018: Nature Protocols
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