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Nature Protocols

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https://www.readbyqxmd.com/read/27906170/generating-high-purity-cardiac-and-endothelial-derivatives-from-patterned-mesoderm-using-human-pluripotent-stem-cells
#1
Nathan J Palpant, Lil Pabon, Clayton E Friedman, Meredith Roberts, Brandon Hadland, Rebecca J Zaunbrecher, Irwin Bernstein, Ying Zheng, Charles E Murry
Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and requires experience in hPSC culture and differentiation techniques. Building on lessons taken from early development, this monolayer-directed differentiation protocol uses different concentrations of activin A and bone morphogenetic protein 4 (BMP4) to polarize cells into mesodermal subtypes that reflect mid-primitive-streak cardiogenic mesoderm and posterior-primitive-streak hemogenic mesoderm...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27906169/automated-identification-of-mouse-visual-areas-with-intrinsic-signal-imaging
#2
Ashley L Juavinett, Ian Nauhaus, Marina E Garrett, Jun Zhuang, Edward M Callaway
Intrinsic signal optical imaging (ISI) is a rapid and noninvasive method for observing brain activity in vivo over a large area of the cortex. Here we describe our protocol for mapping retinotopy to identify mouse visual cortical areas using ISI. First, surgery is performed to attach a head frame to the mouse skull (∼1 h). The next day, intrinsic activity across the visual cortex is recorded during the presentation of a full-field drifting bar in the horizontal and vertical directions (∼2 h). Horizontal and vertical retinotopic maps are generated by analyzing the response of each pixel during the period of the stimulus...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27906168/immunofluorescent-antibody-staining-of-intact-drosophila-larvae
#3
Laurina Manning, Chris Q Doe
Antibody staining is a vital technique for studying the development of many model organisms, including Drosophila. Reliable protocols have long been available for antibody staining of Drosophila whole-mount embryos and dissected larvae. By contrast, methods for staining whole larvae have rarely been reported, are unreliable, and fail to work on large third-instar larvae. This has become a major limitation to understanding the role of multitissue interactions such as neural circuit formation and cell metastasis...
January 2017: Nature Protocols
https://www.readbyqxmd.com/read/27854364/deep-interactome-profiling-of-membrane-proteins-by-co-interacting-protein-identification-technology
#4
Sandra Pankow, Casimir Bamberger, Diego Calzolari, Andreas Bamberger, John R Yates
Affinity purification coupled to mass spectrometry (AP-MS) is the method of choice for analyzing protein-protein interactions, but common protocols frequently recover only the most stable interactions and tend to result in low bait yield for membrane proteins. Here, we present a novel, deep interactome sequencing approach called CoPIT (co-interacting protein identification technology), which allows comprehensive identification and analysis of membrane protein interactomes and their dynamics. CoPIT integrates experimental and computational methods for a coimmunoprecipitation (Co-IP)-based workflow from sample preparation for mass spectrometric analysis to visualization of protein-protein interaction networks...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27854363/indel-variant-analysis-of-short-read-sequencing-data-with-scalpel
#5
Han Fang, Ewa A Bergmann, Kanika Arora, Vladimir Vacic, Michael C Zody, Ivan Iossifov, Jason A O'Rawe, Yiyang Wu, Laura T Jimenez Barron, Julie Rosenbaum, Michael Ronemus, Yoon-Ha Lee, Zihua Wang, Esra Dikoglu, Vaidehi Jobanputra, Gholson J Lyon, Michael Wigler, Michael C Schatz, Giuseppe Narzisi
As the second most common type of variation in the human genome, insertions and deletions (indels) have been linked to many diseases, but the discovery of indels of more than a few bases in size from short-read sequencing data remains challenging. Scalpel (http://scalpel.sourceforge.net) is an open-source software for reliable indel detection based on the microassembly technique. It has been successfully used to discover mutations in novel candidate genes for autism, and it is extensively used in other large-scale studies of human diseases...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27854362/a-bayesian-cluster-analysis-method-for-single-molecule-localization-microscopy-data
#6
Juliette Griffié, Michael Shannon, Claire L Bromley, Lies Boelen, Garth L Burn, David J Williamson, Nicholas A Heard, Andrew P Cope, Dylan M Owen, Patrick Rubin-Delanchy
Cell function is regulated by the spatiotemporal organization of the signaling machinery, and a key facet of this is molecular clustering. Here, we present a protocol for the analysis of clustering in data generated by 2D single-molecule localization microscopy (SMLM)-for example, photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). Three features of such data can cause standard cluster analysis approaches to be ineffective: (i) the data take the form of a list of points rather than a pixel array; (ii) there is a non-negligible unclustered background density of points that must be accounted for; and (iii) each localization has an associated uncertainty in regard to its position...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27831570/a-cell-free-organelle-based-in-vitro-system-for-studying-the-peroxisomal-protein-import-machinery
#7
Tony A Rodrigues, Tânia Francisco, Ana F Dias, Ana G Pedrosa, Cláudia P Grou, Jorge E Azevedo
Here we describe a protocol to dissect the peroxisomal matrix protein import pathway using a cell-free in vitro system. The system relies on a postnuclear supernatant (PNS), which is prepared from rat/mouse liver, to act as a source of peroxisomes and cytosolic components. A typical in vitro assay comprises the following steps: (i) incubation of the PNS with an in vitro-synthesized (35)S-labeled reporter protein; (ii) treatment of the organelle suspension with a protease that degrades reporter proteins that have not associated with peroxisomes; and (iii) SDS-PAGE/autoradiography analysis...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27831569/quantifying-macromolecular-interactions-in-living-cells-using-fret-two-hybrid-assays
#8
Elisabeth S Butz, Manu Ben-Johny, Michael Shen, Philemon S Yang, Lingjie Sang, Martin Biel, David T Yue, Christian Wahl-Schott
Förster resonance energy transfer (FRET) is a versatile method for analyzing protein-protein interactions within living cells. This protocol describes a nondestructive live-cell FRET assay for robust quantification of relative binding affinities for protein-protein interactions. Unlike other approaches, our method correlates the measured FRET efficiencies to relative concentration of interacting proteins to determine binding isotherms while including collisional FRET corrections. We detail how to assemble and calibrate the equipment using experimental and theoretical procedures...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27831568/arqiv-hts-a-versatile-whole-organism-screening-platform-enabling-in-vivo-drug-discovery-at-high-throughput-rates
#9
David T White, Arife Unal Eroglu, Guohua Wang, Liyun Zhang, Sumitra Sengupta, Ding Ding, Surendra K Rajpurohit, Steven L Walker, Hongkai Ji, Jiang Qian, Jeff S Mumm
The zebrafish has emerged as an important model for whole-organism small-molecule screening. However, most zebrafish-based chemical screens have achieved only mid-throughput rates. Here we describe a versatile whole-organism drug discovery platform that can achieve true high-throughput screening (HTS) capacities. This system combines our automated reporter quantification in vivo (ARQiv) system with customized robotics, and is termed 'ARQiv-HTS'. We detail the process of establishing and implementing ARQiv-HTS: (i) assay design and optimization, (ii) calculation of sample size and hit criteria, (iii) large-scale egg production, (iv) automated compound titration, (v) dispensing of embryos into microtiter plates, and (vi) reporter quantification...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27809318/a-high-throughput-functional-genomics-workflow-based-on-crispr-cas9-mediated-targeted-mutagenesis-in-zebrafish
#10
Gaurav K Varshney, Blake Carrington, Wuhong Pei, Kevin Bishop, Zelin Chen, Chunxin Fan, Lisha Xu, Marypat Jones, Matthew C LaFave, Johan Ledin, Raman Sood, Shawn M Burgess
The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27809317/a-microfluidics-based-method-for-measuring-neuronal-activity-in-drosophila-chemosensory-neurons
#11
Lena van Giesen, G Larisa Neagu-Maier, Jae Young Kwon, Simon G Sprecher
Monitoring neuronal responses to defined sensory stimuli is a powerful and widely used approach for understanding sensory coding in the nervous system. However, providing precise, stereotypic and reproducible cues while concomitantly recording neuronal activity remains technically challenging. Here we describe the fabrication and use of a microfluidics system that allows precise temporally restricted stimulation of Drosophila chemosensory neurons with an array of different chemical cues. The system can easily be combined with genetically encoded calcium sensors, and it can measure neuronal activity at single-cell resolution in larval sense organs and in the proboscis or leg of the adult fly...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27809316/the-maxquant-computational-platform-for-mass-spectrometry-based-shotgun-proteomics
#12
Stefka Tyanova, Tikira Temu, Juergen Cox
MaxQuant is one of the most frequently used platforms for mass-spectrometry (MS)-based proteomics data analysis. Since its first release in 2008, it has grown substantially in functionality and can be used in conjunction with more MS platforms. Here we present an updated protocol covering the most important basic computational workflows, including those designed for quantitative label-free proteomics, MS1-level labeling and isobaric labeling techniques. This protocol presents a complete description of the parameters used in MaxQuant, as well as of the configuration options of its integrated search engine, Andromeda...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27809315/rapid-identification-of-causal-mutations-in-tomato-ems-populations-via-mapping-by-sequencing
#13
Virginie Garcia, Cécile Bres, Daniel Just, Lucie Fernandez, Fabienne Wong Jun Tai, Jean-Philippe Mauxion, Marie-Christine Le Paslier, Aurélie Bérard, Dominique Brunel, Koh Aoki, Saleh Alseekh, Alisdair R Fernie, Paul D Fraser, Christophe Rothan
The tomato is the model species of choice for fleshy fruit development and for the Solanaceae family. Ethyl methanesulfonate (EMS) mutants of tomato have already proven their utility for analysis of gene function in plants, leading to improved breeding stocks and superior tomato varieties. However, until recently, the identification of causal mutations that underlie particular phenotypes has been a very lengthy task that many laboratories could not afford because of spatial and technical limitations. Here, we describe a simple protocol for identifying causal mutations in tomato using a mapping-by-sequencing strategy...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27809314/t-rex-on-demand-redox-targeting-in-live-cells
#14
Saba Parvez, Marcus J C Long, Hong-Yu Lin, Yi Zhao, Joseph A Haegele, Vanha N Pham, Dustin K Lee, Yimon Aye
This protocol describes targetable reactive electrophiles and oxidants (T-REX)-a live-cell-based tool designed to (i) interrogate the consequences of specific and time-resolved redox events, and (ii) screen for bona fide redox-sensor targets. A small-molecule toolset comprising photocaged precursors to specific reactive redox signals is constructed such that these inert precursors specifically and irreversibly tag any HaloTag-fused protein of interest (POI) in mammalian and Escherichia coli cells. Syntheses of the alkyne-functionalized endogenous reactive signal 4-hydroxynonenal (HNE(alkyne)) and the HaloTag-targetable photocaged precursor to HNE(alkyne) (also known as Ht-PreHNE or HtPHA) are described...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27809313/large-scale-production-and-isolation-of-candida-biofilm-extracellular-matrix
#15
Robert Zarnowski, Hiram Sanchez, David R Andes
The extracellular matrix of biofilm is unique to the biofilm lifestyle, and it has key roles in community survival. A complete understanding of the biochemical nature of the matrix is integral to the understanding of the roles of matrix components. This knowledge is a first step toward the development of novel therapeutics and diagnostics to address persistent biofilm infections. Many of the assay methods needed for refined matrix composition analysis require milligram amounts of material that is separated from the cellular components of these complex communities...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27809312/labeling-cellular-structures-in-vivo-using-confined-primed-conversion-of-photoconvertible-fluorescent-proteins
#16
Manuel Alexander Mohr, Paul Argast, Periklis Pantazis
The application of green-to-red photoconvertible fluorescent proteins (PCFPs) for in vivo studies in complex 3D tissue structures has remained limited because traditional near-UV photoconversion is not confined in the axial dimension, and photomodulation using axially confined, pulsed near-IR (NIR) lasers has proven inefficient. Confined primed conversion is a dual-wavelength continuous-wave (CW) illumination method that is capable of axially confined green-to-red photoconversion. Here we present a protocol to implement this technique with a commercial confocal laser-scanning microscope (CLSM); evaluate its performance on an in vitro setup; and apply primed conversion for in vivo labeling of single cells in developing zebrafish and mouse preimplantation embryos expressing the green-to-red photoconvertible protein Dendra2...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27809311/laser-microdissection-of-tomato-fruit-cell-and-tissue-types-for-transcriptome-profiling
#17
Laetitia B B Martin, Philippe Nicolas, Antonio J Matas, Yoshihito Shinozaki, Carmen Catalá, Jocelyn K C Rose
This protocol enables transcriptome profiling of specific cell or tissue types that are isolated from tomato using laser microdissection (LM). To prepare tissue for LM, fruit samples are first fixed in optimal cutting temperature (OCT) medium and frozen in molds. The tissue is then sectioned using a cryostat before being dissected using an LM instrument. The RNAs contained in the harvested cells are purified and subjected to two rounds of amplification to yield sufficient quantities of RNA to generate cDNA libraries...
December 2016: Nature Protocols
https://www.readbyqxmd.com/read/27763626/assembly-of-multienzyme-complexes-on-dna-nanostructures
#18
Jinglin Fu, Yuhe Renee Yang, Soma Dhakal, Zhao Zhao, Minghui Liu, Ting Zhang, Nils G Walter, Hao Yan
In nature, the catalytic efficiency of multienzyme complexes highly depends on their spatial organization. The positions and orientations of the composite enzymes are often precisely controlled to facilitate substrate transport between them. Self-assembled DNA nanostructures hold great promise for organizing biomolecules at the nanoscale. Here, we present detailed protocols for exploiting DNA nanostructures as assembly scaffolds that organize the spatial arrangements of multienzyme cascades with control over their relative distance, compartmentalization and substrate diffusion paths...
November 2016: Nature Protocols
https://www.readbyqxmd.com/read/27763625/identification-and-propagation-of-haploid-human-pluripotent-stem-cells
#19
Ido Sagi, Dieter Egli, Nissim Benvenisty
Haploid human pluripotent stem cells (PSCs) integrate haploidy and pluripotency, providing a novel system for functional genomics and developmental research in humans. We have recently derived haploid human embryonic stem cells (ESCs) by parthenogenesis and demonstrated their wide differentiation potential and applicability for genetic screening. Because haploid cells can spontaneously become diploid, their enrichment at an early passage is key for successful derivation. In this protocol, we describe two methodologies, namely metaphase spread analysis and cell sorting, for the identification of haploid human cells within parthenogenetic ESC lines...
November 2016: Nature Protocols
https://www.readbyqxmd.com/read/27763624/preparation-of-plasmonic-vesicles-from-amphiphilic-gold-nanocrystals-grafted-with-polymer-brushes
#20
Jibin Song, Peng Huang, Xiaoyuan Chen
Gold nanovesicles contain multiple nanocrystals within a polymeric coating. The strong plasmonic coupling between adjacent nanoparticles in their vesicular shell makes ultrasensitive biosensing and bioimaging possible. In our laboratory, multifunctional plasmonic vesicles are assembled from amphiphilic gold nanocrystals (such as gold nanoparticles and gold nanorods) coated with mixed hydrophilic and hydrophobic polymer brushes or amphiphilic diblock co-polymer brushes. To fulfill the different requirements of biomedical applications, different polymers that are either pH=responsive, photoactive or biodegradable can be used to form the hydrophobic brush, while the hydrophilicity is maintained by polyethylene glycol (PEG)...
November 2016: Nature Protocols
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