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Nature Protocols

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https://www.readbyqxmd.com/read/28079880/nonradioactive-quantification-of-autophagic-protein-degradation-with-l-azidohomoalanine-labeling
#1
Jigang Wang, Jianbin Zhang, Yew Mun Lee, Shukie Ng, Yin Shi, Zi-Chun Hua, Qingsong Lin, Han-Ming Shen
At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA is used as a surrogate for L-methionine, and, when added to cultured cells grown in methionine-free medium, AHA is incorporated into proteins during de novo protein synthesis. After a chase period to remove short-lived proteins, autophagy is induced by starvation or other stimuli...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/28333915/directed-differentiation-of-human-induced-pluripotent-stem-cells-into-functional-cholangiocyte-like-cells
#2
Fotios Sampaziotis, Miguel Cardoso de Brito, Imbisaat Geti, Alessandro Bertero, Nicholas Rf Hannan, Ludovic Vallier
The difficulty in isolating and propagating functional primary cholangiocytes is a major limitation in the study of biliary disorders and the testing of novel therapeutic agents. To overcome this problem, we have developed a platform for the differentiation of human pluripotent stem cells (hPSCs) into functional cholangiocyte-like cells (CLCs). We have previously reported that our 26-d protocol closely recapitulates key stages of biliary development, starting with the differentiation of hPSCs into endoderm and subsequently into foregut progenitor (FP) cells, followed by the generation of hepatoblasts (HBs), cholangiocyte progenitors (CPs) expressing early biliary markers and mature CLCs displaying cholangiocyte functionality...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28333914/genome-scale-crispr-cas9-knockout-and-transcriptional-activation-screening
#3
Julia Joung, Silvana Konermann, Jonathan S Gootenberg, Omar O Abudayyeh, Randall J Platt, Mark D Brigham, Neville E Sanjana, Feng Zhang
Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28301462/dissection-of-mechanical-force-in-living-cells-by-super-resolved-traction-force-microscopy
#4
Huw Colin-York, Christian Eggeling, Marco Fritzsche
Cells continuously exert or respond to mechanical force. Measurement of these nanoscale forces is a major challenge in cell biology; yet such measurement is essential to the understanding of cell regulation and function. Current methods for examining mechanical force generation either necessitate dedicated equipment or limit themselves to coarse-grained force measurements on the micron scale. In this protocol, we describe stimulated emission depletion traction force microscopy-STED-TFM (STFM), which allows higher sampling of the forces generated by the cell than conventional TFM, leading to a twofold increase in spatial resolution (of up to 500 nm)...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28301461/metabolomics-and-lipidomics-using-traveling-wave-ion-mobility-mass-spectrometry
#5
Giuseppe Paglia, Giuseppe Astarita
Metabolomics and lipidomics aim to profile the wide range of metabolites and lipids that are present in biological samples. Recently, ion mobility spectrometry (IMS) has been used to support metabolomics and lipidomics applications to facilitate the separation and the identification of complex mixtures of analytes. IMS is a gas-phase electrophoretic technique that enables the separation of ions in the gas phase according to their charge, shape and size. Occurring within milliseconds, IMS separation is compatible with modern mass spectrometry (MS) operating with microsecond scan speeds...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28277548/optimized-labeling-of-membrane-proteins-for-applications-to-super-resolution-imaging-in-confined-cellular-environments-using-monomeric-streptavidin
#6
Ingrid Chamma, Olivier Rossier, Grégory Giannone, Olivier Thoumine, Matthieu Sainlos
Recent progress in super-resolution imaging (SRI) has created a strong need to improve protein labeling with probes of small size that minimize the target-to-label distance, increase labeling density, and efficiently penetrate thick biological tissues. This protocol describes a method for labeling genetically modified proteins incorporating a small biotin acceptor peptide with a 3-nm fluorescent probe, monomeric streptavidin. We show how to express, purify, and conjugate the probe to organic dyes with different fluorescent properties, and how to label selectively biotinylated membrane proteins for SRI techniques (point accumulation in nanoscale topography (PAINT), stimulated emission depletion (STED), stochastic optical reconstruction microscopy (STORM))...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28277547/backbone-assignment-of-perdeuterated-proteins-by-solid-state-nmr-using-proton-detection-and-ultrafast-magic-angle-spinning
#7
Pascal Fricke, Veniamin Chevelkov, Maximilian Zinke, Karin Giller, Stefan Becker, Adam Lange
Solid-state NMR (ssNMR) is a technique that allows the study of protein structure and dynamics at atomic detail. In contrast to X-ray crystallography and cryo-electron microscopy, proteins can be studied under physiological conditions-for example, in a lipid bilayer and at room temperature (0-35 °C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years, (1)H-detection of perdeuterated protein samples has been proposed as a method of alleviating the sensitivity issue...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28277546/decerebrate-mouse-model-for-studies-of-the-spinal-cord-circuits
#8
Claire F Meehan, Kyle A Mayr, Marin Manuel, Stan T Nakanishi, Patrick J Whelan
The adult decerebrate mouse model (a mouse with the cerebrum removed) enables the study of sensory-motor integration and motor output from the spinal cord for several hours without compromising these functions with anesthesia. For example, the decerebrate mouse is ideal for examining locomotor behavior using intracellular recording approaches, which would not be possible using current anesthetized preparations. This protocol describes the steps required to achieve a low-blood-loss decerebration in the mouse and approaches for recording signals from spinal cord neurons with a focus on motoneurons...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28253237/translation-complex-profile-sequencing-to-study-the-in-vivo-dynamics-of-mrna-ribosome-interactions-during-translation-initiation-elongation-and-termination
#9
Nikolay E Shirokikh, Stuart K Archer, Traude H Beilharz, David Powell, Thomas Preiss
Messenger RNA (mRNA) translation is a tightly controlled process that is integral to gene expression. It features intricate and dynamic interactions of the small and large subunits of the ribosome with mRNAs, aided by multiple auxiliary factors during distinct initiation, elongation and termination phases. The recently developed ribosome profiling method can generate transcriptome-wide surveys of translation and its regulation. Ribosome profiling records the footprints of fully assembled ribosomes along mRNAs and thus primarily interrogates the elongation phase of translation...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28253236/coordinated-generation-of-multiple-ocular-like-cell-lineages-and-fabrication-of-functional-corneal-epithelial-cell-sheets-from-human-ips-cells
#10
Ryuhei Hayashi, Yuki Ishikawa, Ryousuke Katori, Yuzuru Sasamoto, Yuki Taniwaki, Hiroshi Takayanagi, Motokazu Tsujikawa, Kiyotoshi Sekiguchi, Andrew J Quantock, Kohji Nishida
We describe a protocol for the generation of a functional and transplantable corneal epithelium derived from human induced pluripotent stem (iPS) cells. When this protocol is followed, a proportion of iPS cells spontaneously form circular colonies, each of which is composed of four concentric zones. Cells in these zones have different morphologies and immunostaining characteristics, resembling neuroectoderm, neural crest, ocular-surface ectoderm, or surface ectoderm. We have named this 2D colony a 'SEAM' (self-formed ectodermal autonomous multizone), and previously demonstrated that cells within the SEAM have the potential to give rise to anlages of different ocular lineages, including retinal cells, lens cells, and ocular-surface ectoderm...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28253235/simple-multiplexed-pcr-based-barcoding-of-dna-for-ultrasensitive-mutation-detection-by-next-generation-sequencing
#11
Anders Ståhlberg, Paul M Krzyzanowski, Matthew Egyud, Stefan Filges, Lincoln Stein, Tony E Godfrey
Detection of extremely rare variant alleles within a complex mixture of DNA molecules is becoming increasingly relevant in many areas of clinical and basic research, such as the detection of circulating tumor DNA in the plasma of cancer patients. Barcoding of DNA template molecules early in next-generation sequencing (NGS) library construction provides a way to identify and bioinformatically remove polymerase errors that otherwise make detection of these rare variants very difficult. Several barcoding strategies have been reported, but all require long and complex library preparation protocols...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28253234/engineering-a-humanized-bone-organ-model-in-mice-to-study-bone-metastases
#12
Laure C Martine, Boris M Holzapfel, Jacqui A McGovern, Ferdinand Wagner, Verena M Quent, Parisa Hesami, Felix M Wunner, Cedryck Vaquette, Elena M De-Juan-Pardo, Toby D Brown, Bianca Nowlan, Dan Jing Wu, Cosmo Orlando Hutmacher, Davide Moi, Tatiana Oussenko, Elia Piccinini, Peter W Zandstra, Roberta Mazzieri, Jean-Pierre Lévesque, Paul D Dalton, Anna V Taubenberger, Dietmar W Hutmacher
Current in vivo models for investigating human primary bone tumors and cancer metastasis to the bone rely on the injection of human cancer cells into the mouse skeleton. This approach does not mimic species-specific mechanisms occurring in human diseases and may preclude successful clinical translation. We have developed a protocol to engineer humanized bone within immunodeficient hosts, which can be adapted to study the interactions between human cancer cells and a humanized bone microenvironment in vivo. A researcher trained in the principles of tissue engineering will be able to execute the protocol and yield study results within 4-6 months...
April 2017: Nature Protocols
https://www.readbyqxmd.com/read/28230851/using-hiclip-to-identify-rna-duplexes-that-interact-with-a-specific-rna-binding-protein
#13
Yoichiro Sugimoto, Anob M Chakrabarti, Nicholas M Luscombe, Jernej Ule
The structure of RNA molecules has a critical role in regulating gene expression, largely through influencing their interactions with RNA-binding proteins (RBPs). RNA hybrid and individual-nucleotide resolution UV cross-linking and immunoprecipitation (hiCLIP) is a transcriptome-wide method of monitoring these interactions by identifying RNA duplexes bound by a specific RBP. The hiCLIP protocol consists of the following steps: in vivo cross-linking of RBPs to their bound RNAs; partial RNA digestion and purification of RNA duplexes interacting with the specific RBP using immunoprecipitation; ligation of the two arms of RNA duplexes via a linker; reverse transcription; cDNA library amplification; and finally high-throughput DNA sequencing...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28230850/metal-and-additive-free-photoinduced-borylation-of-haloarenes
#14
Adelphe M Mfuh, Brett D Schneider, Westley Cruces, Oleg V Larionov
Boronic acids and esters have critical roles in the areas of synthetic organic chemistry, molecular sensors, materials science, drug discovery, and catalysis. Many of the current applications of boronic acids and esters require materials with very low levels of transition metal contamination. Most of the current methods for the synthesis of boronic acids, however, require transition metal catalysts and ligands that must be removed via additional purification procedures. This protocol describes a simple, metal- and additive-free method of conversion of haloarenes directly to boronic acids and esters...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28207002/detecting-natural-adaptation-of-the-streptococcus-thermophilus-crispr-cas-systems-in-research-and-classroom-settings
#15
Alexander P Hynes, Marie-Laurence Lemay, Luc Trudel, Hélène Deveau, Michel Frenette, Denise M Tremblay, Sylvain Moineau
CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28207001/genome-editing-using-facs-enrichment-of-nuclease-expressing-cells-and-indel-detection-by-amplicon-analysis
#16
Lindsey A Lonowski, Yoshiki Narimatsu, Anjum Riaz, Catherine E Delay, Zhang Yang, Francesco Niola, Katarzyna Duda, Elke A Ober, Henrik Clausen, Hans H Wandall, Steen H Hansen, Eric P Bennett, Morten Frödin
This protocol describes methods for increasing and evaluating the efficiency of genome editing based on the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated 9) system, transcription activator-like effector nucleases (TALENs) or zinc-finger nucleases (ZFNs). First, Indel Detection by Amplicon Analysis (IDAA) determines the size and frequency of insertions and deletions elicited by nucleases in cells, tissues or embryos through analysis of fluorophore-labeled PCR amplicons covering the nuclease target site by capillary electrophoresis in a sequenator...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28207000/spatial-transcriptomic-analysis-of-cryosectioned-tissue-samples-with-geo-seq
#17
Jun Chen, Shengbao Suo, Patrick Pl Tam, Jing-Dong J Han, Guangdun Peng, Naihe Jing
Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cannot be identified. Here, we describe how to use Geo-seq, a method that combines laser capture microdissection (LCM) and single-cell RNA-seq technology. The combination of these two methods enables the elucidation of cellular heterogeneity and spatial variance simultaneously...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28182019/a-modular-approach-to-the-design-fabrication-and-characterization-of-muscle-powered-biological-machines
#18
Ritu Raman, Caroline Cvetkovic, Rashid Bashir
Biological machines consisting of cells and biomaterials have the potential to dynamically sense, process, respond, and adapt to environmental signals in real time. As a first step toward the realization of such machines, which will require biological actuators that can generate force and perform mechanical work, we have developed a method of manufacturing modular skeletal muscle actuators that can generate up to 1.7 mN (3.2 kPa) of passive tension force and 300 μN (0.56 kPa) of active tension force in response to external stimulation...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28182018/genome-wide-base-resolution-mapping-of-dna-methylation-in-single-cells-using-single-cell-bisulfite-sequencing-scbs-seq
#19
Stephen J Clark, Sébastien A Smallwood, Heather J Lee, Felix Krueger, Wolf Reik, Gavin Kelsey
DNA methylation (DNAme) is an important epigenetic mark in diverse species. Our current understanding of DNAme is based on measurements from bulk cell samples, which obscures intercellular differences and prevents analyses of rare cell types. Thus, the ability to measure DNAme in single cells has the potential to make important contributions to the understanding of several key biological processes, such as embryonic development, disease progression and aging. We have recently reported a method for generating genome-wide DNAme maps from single cells, using single-cell bisulfite sequencing (scBS-seq), allowing the quantitative measurement of DNAme at up to 50% of CpG dinucleotides throughout the mouse genome...
March 2017: Nature Protocols
https://www.readbyqxmd.com/read/28182017/using-connectome-based-predictive-modeling-to-predict-individual-behavior-from-brain-connectivity
#20
Xilin Shen, Emily S Finn, Dustin Scheinost, Monica D Rosenberg, Marvin M Chun, Xenophon Papademetris, R Todd Constable
Neuroimaging is a fast-developing research area in which anatomical and functional images of human brains are collected using techniques such as functional magnetic resonance imaging (fMRI), diffusion tensor imaging (DTI), and electroencephalography (EEG). Technical advances and large-scale data sets have allowed for the development of models capable of predicting individual differences in traits and behavior using brain connectivity measures derived from neuroimaging data. Here, we present connectome-based predictive modeling (CPM), a data-driven protocol for developing predictive models of brain-behavior relationships from connectivity data using cross-validation...
March 2017: Nature Protocols
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