Genome editing of bread wheat using biolistic delivery of CRISPR/Cas9 in vitro transcripts or ribonucleoproteins Nat. Protoc. (IF 12.423) Pub Date : 2018-02-01 Zhen Liang, Kunling Chen, Yi Zhang, Jinxing Liu, Kangquan Yin, Jin-Long Qiu, Caixia Gao
This protocol is an extension to: Nat. Protoc. 9, 2395–2410 (2014); doi:10.1038/nprot.2014.157; published online 18 September 2014
Characterization of homodimer interfaces with cross-linking mass spectrometry and isotopically labeled proteins Nat. Protoc. (IF 12.423) Pub Date : 2018-02-01 Diogo B Lima, John T Melchior, Jamie Morris, Valmir C Barbosa, Julia Chamot-Rooke, Mariana Fioramonte, Tatiana A C B Souza, Juliana S G Fischer, Fabio C Gozzo, Paulo C Carvalho, W Sean Davidson
Cross-linking coupled with mass spectrometry (XL-MS) has emerged as a powerful strategy for the identification of protein–protein interactions, characterization of interaction regions, and obtainment of structural information on proteins and protein complexes. In XL-MS, proteins or complexes are covalently stabilized with cross-linkers and digested, followed by identification of the cross-linked peptides by tandem mass spectrometry (MS/MS). This provides spatial constraints that enable modeling of protein (complex) structures and regions of interaction. However, most XL-MS approaches are not capable of differentiating intramolecular from intermolecular links in multimeric complexes, and therefore they cannot be used to study homodimer interfaces. We have recently developed an approach that overcomes this limitation by stable isotope–labeling of one of the two monomers, thereby creating a homodimer with one 'light' and one 'heavy' monomer. Here, we describe a step-by-step protocol for stable isotope–labeling, followed by controlled denaturation and refolding in the presence of the wild-type protein. The resulting light–heavy dimers are cross-linked, digested, and analyzed by mass spectrometry. We show how to quantitatively analyze the corresponding data with SIM-XL, an XL-MS software with a module tailored toward the MS/MS data from homodimers. In addition, we provide a video tutorial of the data analysis with this protocol. This protocol can be performed in ∼14 d, and requires basic biochemical and mass spectrometry skills.
Investigation of the spatial structure and interactions of the genome at sub-kilobase-pair resolution using T2C Nat. Protoc. (IF 12.423) Pub Date : 2018-02-08 Petros Kolovos, Rutger W W Brouwer, Christel E M Kockx, Michael Lesnussa, Nick Kepper, Jessica Zuin, A M Ali Imam, Harmen J G van de Werken, Kerstin S Wendt, Tobias A Knoch, Wilfred F J van IJcken, Frank Grosveld
Chromosome conformation capture (3C) and its derivatives (e.g., 4C, 5C and Hi-C) are used to analyze the 3D organization of genomes. We recently developed targeted chromatin capture (T2C), an inexpensive method for studying the 3D organization of genomes, interactomes and structural changes associated with gene regulation, the cell cycle, and cell survival and development. Here, we present the protocol for T2C based on capture, describing all experimental steps and bio-informatic tools in full detail. T2C offers high resolution, a large dynamic interaction frequency range and a high signal-to-noise ratio. Its resolution is determined by the resulting fragment size of the chosen restriction enzyme, which can lead to sub-kilobase-pair resolution. T2C's high coverage allows the identification of the interactome of each individual DNA fragment, which makes binning of reads (often used in other methods) basically unnecessary. Notably, T2C requires low sequencing efforts. T2C also allows multiplexing of samples for the direct comparison of multiple samples. It can be used to study topologically associating domains (TADs), determining their position, shape, boundaries, and intra- and inter-domain interactions, as well as the composition of aggregated loops, interactions between nucleosomes, individual transcription factor binding sites, and promoters and enhancers. T2C can be performed by any investigator with basic skills in molecular biology techniques in ∼7–8 d. Data analysis requires basic expertise in bioinformatics and in Linux and Python environments.
Structural prediction of protein models using distance restraints derived from cross-linking mass spectrometry data Nat. Protoc. (IF 12.423) Pub Date : 2018-02-08 Zsuzsanna Orbán-Németh, Rebecca Beveridge, David M Hollenstein, Evelyn Rampler, Thomas Stranzl, Otto Hudecz, Johannes Doblmann, Peter Schlögelhofer, Karl Mechtler
This protocol describes a workflow for creating structural models of proteins or protein complexes using distance restraints derived from cross-linking mass spectrometry experiments. The distance restraints are used (i) to adjust preliminary models that are calculated on the basis of a homologous template and primary sequence, and (ii) to select the model that is in best agreement with the experimental data. In the case of protein complexes, the cross-linking data are further used to dock the subunits to one another to generate models of the interacting proteins. Predicting models in such a manner has the potential to indicate multiple conformations and dynamic changes that occur in solution. This modeling protocol is compatible with many cross-linking workflows and uses open-source programs or programs that are free for academic users and do not require expertise in computational modeling. This protocol is an excellent additional application with which to use cross-linking results for building structural models of proteins. The established protocol is expected to take 6–12 d to complete, depending on the size of the proteins and the complexity of the cross-linking data.
Production of ready-to-use few-layer graphene in aqueous suspensions Nat. Protoc. (IF 12.423) Pub Date : 2018-02-15 Jose M González-Domínguez, Verónica León, María Isabel Lucío, Maurizio Prato, Ester Vázquez
Graphene has promising physical and chemical properties such as high strength and flexibility, coupled with high electrical and thermal conductivities. It is therefore being incorporated into polymer-based composites for use in electronics and photonics applications. A main constraint related to the graphene development is that, being of a strongly hydrophobic nature, almost all dispersions (usually required for its handling and processing toward the desired application) are prepared in poisonous organic solvents such as N-methyl pyrrolidone or N,N-dimethyl formamide. Here, we describe how to prepare exfoliated graphite using a ball mill. The graphene produced is three to four layers thick and ∼500 nm in diameter on average, as measured by electron microscopy and Raman spectroscopy; can be stored in the form of light solid; and is easily dispersed in aqueous media. Our methodology consists of four main steps: (i) the mechanochemical intercalation of organic molecules (melamine) into graphite, followed by suspension in water; (ii) the washing of suspended graphene to eliminate most of the melamine; (iii) the isolation of stable graphene sheets; and (iv) freeze–drying to obtain graphene powder. This process takes 6–7 or 9–10 d for aqueous suspensions and dry powders, respectively. The product has well-defined properties and can be used for many science and technology applications, including toxicology impact assessment and the production of innovative medical devices.
An experimental toolbox for characterization of mammalian collagen type I in biological specimens Nat. Protoc. (IF 12.423) Pub Date : 2018-02-15 Héctor Capella-Monsonís, João Q Coentro, Valeria Graceffa, Zhuning Wu, Dimitrios I Zeugolis
Collagen type I is the most abundant extracellular matrix protein, and collagen type I supramolecular assemblies (e.g., tissue grafts, biomaterials and cell-assembled systems) are used extensively in tissue engineering and regenerative medicine. Many studies, for convenience or economic reasons, do not accurately determine collagen type I purity, concentration, solubility and extent of cross-linking in biological specimens, frequently resulting in erroneous conclusions. In this protocol, we describe solubility; normal, reduced and delayed (interrupted) SDS-PAGE; hydroxyproline; Sircol collagen and Pierce BCA protein; denaturation temperature; ninhydrin/trinitrobenzene sulfonic acid; and collagenase assays and assess them in a diverse range of biological samples (e.g., tissue samples; purified solutions or lyophilized materials; 3D scaffolds, such as sponges and hydrogels; and cell media and layers). Collectively, the described protocols provide a comprehensive, yet fast and readily implemented, toolbox for collagen type I characterization in any biological specimen.
Proteome-wide identification of ubiquitin interactions using UbIA-MS Nat. Protoc. (IF 12.423) Pub Date : 2018-02-15 Xiaofei Zhang, Arne H Smits, Gabrielle BA van Tilburg, Huib Ovaa, Wolfgang Huber, Michiel Vermeulen
Ubiquitin-binding proteins play an important role in eukaryotes by translating differently linked polyubiquitin chains into proper cellular responses. Current knowledge about ubiquitin-binding proteins and ubiquitin linkage-selective interactions is mostly based on case-by-case studies. We have recently reported a method called ubiquitin interactor affinity enrichment–mass spectrometry (UbIA-MS), which enables comprehensive identification of ubiquitin interactors for all ubiquitin linkages from crude cell lysates. One major strength of UbIA-MS is the fact that ubiquitin interactors are enriched from crude cell lysates, in which proteins are present at endogenous levels, contain biologically relevant post-translational modifications (PTMs) and are assembled in native protein complexes. In addition, UbIA-MS uses chemically synthesized nonhydrolyzable diubiquitin, which mimics native diubiquitin and is inert to cleavage by endogenous deubiquitinases (DUBs). Here, we present a detailed protocol for UbIA-MS that proceeds in five stages: (i) chemical synthesis of ubiquitin precursors and click chemistry for the generation of biotinylated nonhydrolyzable diubiquitin baits, (ii) in vitro affinity purification of ubiquitin interactors, (iii) on-bead interactor digestion, (iv) liquid chromatography (LC)–MS/MS analysis and (v) data analysis to identify differentially enriched proteins. The computational analysis tools are freely available as an open-source R software package, including a graphical interface. Typically, UbIA-MS allows the identification of dozens to hundreds of ubiquitin interactors from any type of cell lysate, and can be used to study cell type or stimulus-dependent ubiquitin interactions. The nonhydrolyzable diubiquitin synthesis can be completed in 3 weeks, followed by ubiquitin interactor enrichment and identification, which can be completed within another 2 weeks.
Genome-wide mapping of endogenous G-quadruplex DNA structures by chromatin immunoprecipitation and high-throughput sequencing Nat. Protoc. (IF 12.423) Pub Date : 2018-02-22 Robert Hänsel-Hertsch, Jochen Spiegel, Giovanni Marsico, David Tannahill, Shankar Balasubramanian
G-rich DNA sequences can form four-stranded G-quadruplex (G4) secondary structures and are linked to fundamental biological processes such as transcription, replication and telomere maintenance. G4s are also implicated in promoting genome instability, cancer and other diseases. Here, we describe a detailed G4 ChIP-seq method that robustly enables the determination of G4 structure formation genome-wide in chromatin. This protocol adapts traditional ChIP-seq for the detection of DNA secondary structures through the use of a G4-structure-specific single-chain antibody with refinements in chromatin immunoprecipitation followed by high-throughput sequencing. This technology does not require expression of the G4 antibody in situ, enabling broad applicability to theoretically all chromatin sources. Beginning with chromatin isolation and antibody preparation, the entire protocol can be completed in <1 week, including basic computational analysis.
Generation of human brain region–specific organoids using a miniaturized spinning bioreactor Nat. Protoc. (IF 12.423) Pub Date : 2018-02-22 Xuyu Qian, Fadi Jacob, Mingxi Max Song, Ha Nam Nguyen, Hongjun Song, Guo-li Ming
Human brain organoids, 3D self-assembled neural tissues derived from pluripotent stem cells, are important tools for studying human brain development and related disorders. Suspension cultures maintained by spinning bioreactors allow for the growth of large organoids despite the lack of vasculature, but commercially available spinning bioreactors are bulky in size and have low throughput. Here, we describe the procedures for building the miniaturized multiwell spinning bioreactor SpinΩ from 3D-printed parts and commercially available hardware. We also describe how to use SpinΩ to generate forebrain, midbrain and hypothalamus organoids from human induced pluripotent stem cells (hiPSCs). These organoids recapitulate key dynamic features of the developing human brain at the molecular, cellular and structural levels. The reduction in culture volume, increase in throughput and reproducibility achieved using our bioreactor and region-specific differentiation protocols enable quantitative modeling of brain disorders and compound testing. This protocol takes 14–84 d to complete (depending on the type of brain region–specific organoids and desired developmental stages), and organoids can be further maintained over 200 d. Competence with hiPSC culture is required for optimal results.
Monitoring intracellular nanomolar calcium using fluorescence lifetime imaging Nat. Protoc. (IF 12.423) Pub Date : 2018-02-22 Kaiyu Zheng, Thomas P Jensen, Dmitri A Rusakov
Nanomolar-range fluctuations of intracellular [Ca2+] are critical for brain cell function but remain difficult to measure. We have advanced a microscopy technique to monitor intracellular [Ca2+] in individual cells in acute brain slices (also applicable in vivo) using fluorescence lifetime imaging (FLIM) of the Ca2+-sensitive fluorescent indicator Oregon Green BAPTA1 (OGB-1). The OGB-1 fluorescence lifetime is sensitive to [Ca2+] within the 10–500 nM range but not to other factors such as viscosity, temperature, or pH. This protocol describes the requirements, setup, and calibration of the FLIM system required for OGB-1 imaging. We provide a step-by-step procedure for whole-cell OGB-1 loading and two-photon FLIM. We also describe how to analyze the obtained FLIM data using total photon count and gated-intensity record, a ratiometric photon-counting approach that provides a highly improved signal-to-noise ratio and greater sensitivity of absolute [Ca2+] readout. We demonstrate our technique in nerve cells in situ, and it is adaptable to other cell types and fluorescent indicators. This protocol requires a basic understanding of FLIM and experience in single-cell electrophysiology and cell imaging. Setting up the FLIM system takes ∼2 d, and OGB-1 loading, imaging, and data analysis take 2 d.
Promoting the accumulation of tumor-specific T cells in tumor tissues by dendritic cell vaccines and chemokine-modulating agents Nat. Protoc. (IF 12.423) Pub Date : 2018-01-18 Nataša Obermajer, Julie Urban, Eva Wieckowski, Ravikumar Muthuswamy, Roshni Ravindranathan, David L Bartlett, Pawel Kalinski
This protocol describes how to induce large numbers of tumor-specific cytotoxic T cells (CTLs) in the spleens and lymph nodes of mice receiving dendritic cell (DC) vaccines and how to modulate tumor microenvironments (TMEs) to ensure effective homing of the vaccination-induced CTLs to tumor tissues. We also describe how to evaluate the numbers of tumor-specific CTLs within tumors. The protocol contains detailed information describing how to generate a specialized DC vaccine with augmented ability to induce tumor-specific CTLs. We also describe methods to modulate the production of chemokines in the TME and show how to quantify tumor-specific CTLs in the lymphoid organs and tumor tissues of mice receiving different treatments. The combined experimental procedure, including tumor implantation, DC vaccine generation, chemokine-modulating (CKM) approaches, and the analyses of tumor-specific systemic and intratumoral immunity is performed over 30–40 d. The presented ELISpot-based ex vivo CTL assay takes 6 h to set up and 5 h to develop. In contrast to other methods of evaluating tumor-specific immunity in tumor tissues, our approach allows detection of intratumoral T-cell responses to nonmanipulated weakly immunogenic cancers. This detection method can be performed using basic laboratory skills, and facilitates the development and preclinical evaluation of new immunotherapies.
CRISPR/Cas9 genome editing in human hematopoietic stem cells Nat. Protoc. (IF 12.423) Pub Date : 2018-01-25 Rasmus O Bak, Daniel P Dever, Matthew H Porteus
Genome editing via homologous recombination (HR) (gene targeting) in human hematopoietic stem cells (HSCs) has the power to reveal gene–function relationships and potentially transform curative hematological gene and cell therapies. However, there are no comprehensive and reproducible protocols for targeting HSCs for HR. Herein, we provide a detailed protocol for the production, enrichment, and in vitro and in vivo analyses of HR-targeted HSCs by combining CRISPR/Cas9 technology with the use of rAAV6 and flow cytometry. Using this protocol, researchers can introduce single-nucleotide changes into the genome or longer gene cassettes with the precision of genome editing. Along with our troubleshooting and optimization guidelines, researchers can use this protocol to streamline HSC genome editing at any locus of interest. The in vitro HSC-targeting protocol and analyses can be completed in 3 weeks, and the long-term in vivo HSC engraftment analyses in immunodeficient mice can be achieved in 16 weeks. This protocol enables manipulation of genes for investigation of gene functions during hematopoiesis, as well as for the correction of genetic mutations in HSC transplantation–based therapies for diseases such as sickle cell disease, β-thalassemia, and primary immunodeficiencies.
Mass cytometry analysis of immune cells in the brain Nat. Protoc. (IF 12.423) Pub Date : 2018-01-25 Ben Korin, Tania Dubovik, Asya Rolls
This protocol describes how to characterize immune cells in the mouse brain compartment using mass cytometry (CyTOF) and provides a step-by-step description from brain dissection to data analysis.
Heat-induced radiolabeling and fluorescence labeling of Feraheme nanoparticles for PET/SPECT imaging and flow cytometry Nat. Protoc. (IF 12.423) Pub Date : 2018-01-25 Hushan Yuan, Moses Q Wilks, Marc D Normandin, Georges El Fakhri, Charalambos Kaittanis, Lee Josephson
This protocol describes the heat-induced radiolabeling of Feraheme (FH) nanoparticles for PET/SPECT imaging and labeling of FH nanoparticles with fluorescent Cy5.5 dyes for flow cytometry.
Multiplexed proteome analysis with neutron-encoded stable isotope labeling in cells and mice Nat. Protoc. (IF 12.423) Pub Date : 2018-01-11 Katherine A Overmyer, Stefka Tyanova, Alex S Hebert, Michael S Westphall, Jürgen Cox, Joshua J Coon
We describe a protocol for multiplexed proteomic analysis using neutron-encoded (NeuCode) stable isotope labeling of amino acids in cells (SILAC) or mice (SILAM). This method currently enables simultaneous comparison of up to nine treatment and control proteomes. Another important advantage over traditional SILAC/SILAM is that shorter labeling times are required. Exploiting the small mass differences that correspond to subtle differences in the neutron-binding energies of different isotopes, the amino acids used in NeuCode SILAC/SILAM differ in mass by just a few milliDaltons. Isotopologs of lysine are introduced into cells or mammals, via the culture medium or diet, respectively, to metabolically label the proteome. Labeling time is ∼2 weeks for cultured cells and 3–4 weeks for mammals. The proteins are then extracted, relevant samples are combined, and these are enzymatically digested with lysyl endopeptidase (Lys-C). The resultant peptides are chromatographically separated and then mass analyzed. During mass spectrometry (MS) data acquisition, high-resolution MS1 spectra (≥240,000 resolving power at m/z = 400) reveal the embedded isotopic signatures, enabling relative quantification, while tandem mass spectra, collected at lower resolutions, provide peptide identities. Both types of spectra are processed using NeuCode-enabled MaxQuant software. In total, the approximate completion time for the protocol is 3–5 weeks.
CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii Nat. Protoc. (IF 12.423) Pub Date : 2018-01-11 Saima M Sidik, Diego Huet, Sebastian Lourido
Apicomplexan parasites, such as Toxoplasma gondii, cause extensive morbidity and mortality in humans and livestock, highlighting the need for a deeper understanding of their molecular biology. Although techniques for the generation of targeted gene disruptions have long been available for apicomplexans, such methods are not readily scalable to the entire genome. We recently used CRISPR-Cas9 to disrupt all nuclear protein–coding genes in T. gondii using a pooled format. The method relies on transfection of a guide RNA library into parasites constitutively expressing Cas9. Here, we present the complete workflow of such a screen, including preparation of the guide RNA library, growth and testing of the recipient strain, generation of the mutant population, culture conditions for the screen, preparation of genomic DNA libraries, next-generation sequencing of the guide RNA loci, and analysis to detect fitness-conferring genes. This method can be deployed to study how culture conditions affect the repertoire of genes needed by parasites, which will enable studies of their metabolic needs, host specificity, and drug-resistance mechanisms. In addition, by manipulating the background in which the screen is performed, researchers will be able to investigate genetic interactions, which may help uncover redundancy or epistasis in the parasite genome. Using this method, a genome-wide screen and its analysis can be completed in 3 weeks, after ∼1 month of preparation to generate the library and grow the cells needed, making it a powerful tool for uncovering functionally important genes in apicomplexan parasites.
On-demand synthesis of organozinc halides under continuous flow conditions Nat. Protoc. (IF 12.423) Pub Date : 2018-01-11 Mateo Berton, Lena Huck, Jesús Alcázar
Organozinc reagents are versatile building blocks for introducing C(sp2)-C(sp3) and C(sp3)-C(sp3) bonds into organic structures. However, despite their ample synthetic versatility and broad functional group tolerance, the use of organozinc reagents in the laboratory is limited because of their instability, exothermicity and water sensitivity, as well as their labor-intensive preparation. Herein, we describe an on-demand synthesis of these useful reagents under continuous flow conditions, overcoming these primary limitations and supporting widespread adoption of these reagents in synthetic organic chemistry. To exemplify this procedure, a solution of ethyl zincbromoacetate is prepared by flowing ethyl bromoacetate through a column containing metallic zinc. The temperature of the column is controlled by a heating jacket and a thermocouple in close contact with it. Advice on how to perform the procedure using alternative equipment is also given to allow a wider access to the methodology. Here we describe the preparation of 50 ml of solution, which takes 1 h 40 min, although up to 250–300 ml can be prepared with the same column setup at a rate of 30 ml per h. The procedure provides the reagent as a clean solution with reproducible concentration. Organozinc solutions generated in flow can be coupled to a second flow reactor to perform a Reformatsky reaction or can be collected over a flask containing the required reagents for a batch Negishi reaction.
Colonoscopy-based colorectal cancer modeling in mice with CRISPR–Cas9 genome editing and organoid transplantation Nat. Protoc. (IF 12.423) Pub Date : 2018-01-04 Jatin Roper, Tuomas Tammela, Adam Akkad, Mohammad Almeqdadi, Sebastian B Santos, Tyler Jacks, Ömer H Yilmaz
Most genetically engineered mouse models (GEMMs) of colorectal cancer are limited by tumor formation in the small intestine, a high tumor burden that limits metastasis, and the need to generate and cross mutant mice. Cell line or organoid transplantation models generally produce tumors in ectopic locations—such as the subcutaneous space, kidney capsule, or cecal wall—that do not reflect the native stromal environment of the colon mucosa. Here, we describe detailed protocols to rapidly and efficiently induce site-directed tumors in the distal colon of mice that are based on colonoscopy-guided mucosal injection. These techniques can be adapted to deliver viral vectors carrying Cre recombinase, CRISPR–Cas9 components, CRISPR-engineered mouse tumor organoids, or human cancer organoids to mice to model the adenoma–carcinoma–metastasis sequence of tumor progression. The colonoscopy injection procedure takes ∼15 min, including preparation. In our experience, anyone with reasonable hand–eye coordination can become proficient with mouse colonoscopy and mucosal injection with a few hours of practice. These approaches are ideal for a wide range of applications, including assessment of gene function in tumorigenesis, examination of tumor–stroma interactions, studies of cancer metastasis, and translational research with patient-derived cancers.
A surgical orthotopic organoid transplantation approach in mice to visualize and study colorectal cancer progression Nat. Protoc. (IF 12.423) Pub Date : 2018-01-04 Arianna Fumagalli, Saskia J E Suijkerbuijk, Harry Begthel, Evelyne Beerling, Koen C Oost, Hugo J Snippert, Jacco van Rheenen, Jarno Drost
Most currently available colorectal cancer (CRC) mouse models are not suitable for studying progression toward the metastatic stage. Recently, establishment of tumor organoid lines, either from murine CRC models or patients, and the possibility of engineering them with genome-editing technologies, have provided a large collection of tumor material faithfully recapitulating phenotypic and genetic heterogeneity of native tumors. To study tumor progression in the natural in vivo environment, we developed an orthotopic approach based on transplantation of CRC organoids into the cecal epithelium. The 20-min procedure is described in detail here and enables growth of transplanted organoids into a single tumor mass within the intestinal tract. Due to long latency, tumor cells are capable of spreading through the blood circulation and forming metastases at distant sites. This method is designed to generate tumors suitable for studying CRC progression, thereby providing the opportunity to visualize tumor cell dynamics in vivo in real time by intravital microscopy.
Use of a three-layer gradient system of cells for rat testicular organoid generation Nat. Protoc. (IF 12.423) Pub Date : 2018-01-04 João Pedro Alves-Lopes, Olle Söder, Jan-Bernd Stukenborg
We have recently developed a 3D culture system that allows the reorganization of rat primary testicular cells into organoids with a functional blood–testis barrier, as well as the establishment and maintenance of germ cells. The innovative aspect of our model, the three-layer gradient system (3-LGS), comprises cells combined with Matrigel placed between two layers of Matrigel without cells, which creates a gradient of cells and allows the reorganization of testicular cells into organized structures after 5–7 d in culture. This reorganization is not observed when testicular cells are suspended in only one layer of Matrigel, the methodology used in the majority of the protocols for generating organoids. The model can be applied to follow and quantify cell migration during testicular organoid formation, and to explore the role of growth factors and the toxic effects of drugs and environmental contaminants on germ cell maintenance and blood–testis barrier integrity. The 3-LGS is a robust and reproducible method that requires a small volume of Matrigel and a low number of cells (16 μl and 132,000 cells, respectively), enabling and facilitating high-throughput analysis of germ-to-somatic cell associations in vitro.
High-throughput in situ X-ray screening of and data collection from protein crystals at room temperature and under cryogenic conditions Nat. Protoc. (IF 12.423) Pub Date : 2018-01-04 Jana Broecker, Takefumi Morizumi, Wei-Lin Ou, Viviane Klingel, Anling Kuo, David J Kissick, Andrii Ishchenko, Ming-Yue Lee, Shenglan Xu, Oleg Makarov, Vadim Cherezov, Craig M Ogata, Oliver P Ernst
Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20–25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min.
3D molecular cartography using LC–MS facilitated by Optimus and 'ili software Nat. Protoc. (IF 12.423) Pub Date : 2017-12-21 Ivan Protsyuk, Alexey V Melnik, Louis-Felix Nothias, Luca Rappez, Prasad Phapale, Alexander A Aksenov, Amina Bouslimani, Sergey Ryazanov, Pieter C Dorrestein, Theodore Alexandrov
Our skin, our belongings, the world surrounding us, and the environment we live in are covered with molecular traces. Detecting and characterizing these molecular traces is necessary to understand the environmental impact on human health and disease, and to decipher complex molecular interactions between humans and other species, particularly microbiota. We recently introduced 3D molecular cartography for mapping small organic molecules (including metabolites, lipids, and environmental molecules) found on various surfaces, including the human body. Here, we provide a protocol and open-source software for 3D molecular cartography. The protocol includes step-by-step procedures for sample collection and processing, liquid chromatography–mass spectrometry (LC–MS)-based metabolomics, quality control (QC), molecular identification using MS/MS, data processing, and visualization with 3D models of the sampled environment. The LC–MS method was optimized for a broad range of small organic molecules. We enable scientists to reproduce our previously obtained results, and illustrate the broad utility of our approach with molecular maps of a rosemary plant and an ATM keypad after a PIN code was entered. To promote reproducibility, we introduce cartographical snapshots: files that describe a particular map and visualization settings, and that can be shared and loaded to reproduce the visualization. The protocol enables molecular cartography to be performed in any mass spectrometry laboratory and, in principle, for any spatially mapped data. We anticipate applications, in particular, in medicine, ecology, agriculture, biotechnology, and forensics. The protocol takes 78 h for a molecular map of 100 spots, excluding the reagent setup.
Live-cell measurements of kinase activity in single cells using translocation reporters Nat. Protoc. (IF 12.423) Pub Date : 2017-12-21 Takamasa Kudo, Stevan Jeknić, Derek N Macklin, Sajia Akhter, Jacob J Hughey, Sergi Regot, Markus W Covert
Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. We have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization. The negative charge introduced by phosphorylation of the substrate is used to directly modulate nuclear import and export, thereby regulating the reporter's distribution between the cytoplasm and nucleus. The relative cytoplasmic versus nuclear fluorescence of the KTR construct (the C/N ratio) is used as a proxy for the kinase activity in living, single cells. Multiple KTRs can be studied in the same cell by fusing them to different fluorescent proteins. Here, we present a protocol to execute and analyze live-cell microscopy experiments using KTRs. We describe strategies for development of new KTRs and procedures for lentiviral expression of KTRs in a cell line of choice. Cells are then plated in a 96-well plate, from which multichannel fluorescent images are acquired with automated time-lapse microscopy. We provide detailed guidance for a computational analysis and parameterization pipeline. The entire procedure, from virus production to data analysis, can be completed in ∼10 d.
Single-cell microscopy of suspension cultures using a microfluidics-assisted cell screening platform Nat. Protoc. (IF 12.423) Pub Date : 2017-12-21 Burak Okumus, Charles J Baker, Juan Carlos Arias-Castro, Ghee Chuan Lai, Emanuele Leoncini, Somenath Bakshi, Scott Luro, Dirk Landgraf, Johan Paulsson
Studies that rely on fluorescence imaging of nonadherent cells that are cultured in suspension, such as Escherichia coli, are often hampered by trade-offs that must be made between data throughput and imaging resolution. We developed a platform for microfluidics-assisted cell screening (MACS) that overcomes this trade-off by temporarily immobilizing suspension cells within a microfluidics chip. This enables high-throughput and automated single-cell microscopy for a wide range of cell types and sizes. As cells can be rapidly sampled directly from a suspension culture, MACS bypasses the need for sample preparation, and therefore allows measurements without perturbing the native cell physiology. The setup can also be integrated with complex growth chambers, and can be used to enrich or sort the imaged cells. Furthermore, MACS facilitates the visualization of individual cytoplasmic fluorescent proteins (FPs) in E. coli, allowing low-abundance proteins to be counted using standard total internal reflection fluorescence (TIRF) microscopy. Finally, MACS can be used to impart mechanical pressure for assessing the structural integrity of individual cells and their response to mechanical perturbations, or to make cells take up chemicals that otherwise would not pass through the membrane. This protocol describes the assembly of electronic control circuitry, the construction of liquid-handling components and the creation of the MACS microfluidics chip. The operation of MACS is described, and automation software is provided to integrate MACS control with image acquisition. Finally, we provide instructions for extending MACS using an external growth chamber (1 d) and for how to sort rare cells of interest.
Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors Nat. Protoc. (IF 12.423) Pub Date : 2017-12-21 Hiromi Miura, Rolen M Quadros, Channabasavaiah B Gurumurthy, Masato Ohtsuka
CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1–10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts–CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30–60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.
Transient expression of human antibodies in mammalian cells Nat. Protoc. (IF 12.423) Pub Date : 2017-12-14 Rodrigo Vazquez-Lombardi, Damien Nevoltris, Ansha Luthra, Peter Schofield, Carsten Zimmermann, Daniel Christ
Mammalian cells are powerful expression systems for producing glycosylated recombinant antibody preparations with minimal endotoxin contamination. This protocol describes procedures for antibody design, expression, purification and characterization.
Use of TAI-FISH to visualize neural ensembles activated by multiple stimuli Nat. Protoc. (IF 12.423) Pub Date : 2017-12-14 Qi Zhang, Qiye He, Jihua Wang, Chaoying Fu, Hailan Hu
This protocol describes a dual mRNA and protein labeling strategy that allows identification of activated neuronal assemblies in response to two temporally separated stimuli in mouse brain sections.
Mapping the small RNA interactome in bacteria using RIL-seq Nat. Protoc. (IF 12.423) Pub Date : 2017-12-07 Sahar Melamed, Raya Faigenbaum-Romm, Asaf Peer, Niv Reiss, Omer Shechter, Amir Bar, Yael Altuvia, Liron Argaman, Hanah Margalit
This protocol describes an experimental–computational methodology for mapping the small RNA interactome in bacteria.
Expansion of patient-derived circulating tumor cells from liquid biopsies using a CTC microfluidic culture device Nat. Protoc. (IF 12.423) Pub Date : 2017-12-07 Bee Luan Khoo, Gianluca Grenci, Ying Bena Lim, Soo Chin Lee, Jongyoon Han, Chwee Teck Lim
This protocol describes a microfluidics approach for culturing liquid-biopsy-derived circulating tumor cell clusters to predict a patient's response toward various therapeutic strategies.
Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR Nat. Protoc. (IF 12.423) Pub Date : 2017-12-07 Franz Hagn, Mahmoud L Nasr, Gerhard Wagner
The applications of solution-state NMR of membrane proteins are often limited by difficulty in finding a suitable membrane mimetic of tailored size that shows native-like membrane properties and provides long-term stability. This protocol describes how to assemble phospholipid nanodiscs and incorporate membrane proteins for NMR-structural studies.
Measuring mutation accumulation in single human adult stem cells by whole-genome sequencing of organoid cultures Nat. Protoc. (IF 12.423) Pub Date : 2017-12-07 Myrthe Jager, Francis Blokzijl, Valentina Sasselli, Sander Boymans, Roel Janssen, Nicolle Besselink, Hans Clevers, Ruben van Boxtel, Edwin Cuppen
This protocol describes a method for cataloging genome-wide mutations that accumulated during life or culture in single adult stem cells of different human tissues, by combining whole-genome sequencing with organoid-culture technologies.
Langmuir–Blodgett nanotemplates for protein crystallography Nat. Protoc. (IF 12.423) Pub Date : 2017-11-30 Eugenia Pechkova, Claudio Nicolini
Protein crystallization still presents a challenge for X-ray crystallography. This protocol describes the Langmuir–Blodgett nanotemplate method, in which 2D protein LB nanotemplates trigger formation of 3D protein crystals by hanging-drop vapor diffusion.
Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules Nat. Protoc. (IF 12.423) Pub Date : 2017-11-30 Merja Joensuu, Ramon Martínez-Mármol, Pranesh Padmanabhan, Nick R Glass, Nela Durisic, Matthew Pelekanos, Mahdie Mollazade, Giuseppe Balistreri, Rumelo Amor, Justin J Cooper-White, Geoffrey J Goodhill, Frédéric A Meunier
This protocol describes a pulse–chase approach to studying activity-dependent internalization of fluorescent ligands into endocytic compartments using subdiffractional single-particle tracking in live hippocampal neurons.
Preparation of viable adult ventricular myocardial slices from large and small mammals Nat. Protoc. (IF 12.423) Pub Date : 2017-11-30 Samuel A Watson, Martina Scigliano, Ifigeneia Bardi, Raimondo Ascione, Cesare M Terracciano, Filippo Perbellini
This protocol describes how to obtain 100- to 400-μm-thick slices of a living myocardium from rodents, pigs, humans and dogs that retain the native multicellularity, architecture and physiology of the heart.
Deriving genotypes from RAD-seq short-read data using Stacks Nat. Protoc. (IF 12.423) Pub Date : 2017-11-30 Nicolas C Rochette, Julian M Catchen
In this protocol, the authors provide a strategy and set of methods to analyze restriction-site-associated DNA-sequencing (RAD-seq) data using Stacks, enabling the genome-wide discovery and genotyping of SNPs across a range of systems.
Production of knock-in mice in a single generation from embryonic stem cells Nat. Protoc. (IF 12.423) Pub Date : 2017-11-16 Hideki Ukai, Hiroshi Kiyonari, Hiroki R Ueda
This protocol describes the generation of mice entirely derived from genome-edited embryonic stem cells, enabling the production of transgenic mice in a single generation.
Multimodal profiling of single-cell morphology, electrophysiology, and gene expression using Patch-seq Nat. Protoc. (IF 12.423) Pub Date : 2017-11-16 Cathryn R Cadwell, Federico Scala, Shuang Li, Giulia Livrizzi, Shan Shen, Rickard Sandberg, Xiaolong Jiang, Andreas S Tolias
This protocol describes how to integrate whole-cell patch-clamp in single neurons from mouse brain tissue slices with single-cell RNA sequencing and morphological recovery.
Chemical synthesis of membrane proteins by the removable backbone modification method Nat. Protoc. (IF 12.423) Pub Date : 2017-11-16 Shan Tang, Chao Zuo, Dong-Liang Huang, Xiao-Ying Cai, Long-Hua Zhang, Chang-Lin Tian, Ji-Shen Zheng, Lei Liu
This protocol describes how to chemically synthesize membrane proteins through the installation of solubilizing removable backbone modification tags into hydrophobic transmembrane peptides. The implementation of the protocol is demonstrated by the chemical synthesis of phosphorylated M2 (M2-pSer64), a 97-aa proton channel protein from the influenza A virus. The synthesis of M2-pSer64 at milligram scale takes ∼200 working hours (excluding the time for lyophilizations).
Compartmentalized partnered replication for the directed evolution of genetic parts and circuits Nat. Protoc. (IF 12.423) Pub Date : 2017-11-09 Zhanar Abil, Jared W Ellefson, Jimmy D Gollihar, Ella Watkins, Andrew D Ellington
This protocol describes the procedures for compartmentalized partnered replication (CPR), an emulsion-based directed evolution method for the generation of proteins, genetic elements, and genetic circuits with improved or altered function.
Chromatin-state discovery and genome annotation with ChromHMM Nat. Protoc. (IF 12.423) Pub Date : 2017-11-09 Jason Ernst, Manolis Kellis
This protocol describes how to use ChromHMM, a robust open-source software package that enables the learning of chromatin states, annotates their occurrences across the genome, and facilitates their biological interpretation.
Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap Nat. Protoc. (IF 12.423) Pub Date : 2017-11-02 Alfredo Castello, Christian K. Frese, Bernd Fischer, Aino I Järvelin, Rastislav Horos, Anne-Marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W Hentze
Here the authors provide an extension to their earlier RNA interactome capture protocol. This Protocol Extension describes RBDmap—a method to identify the regions of RNA-binding proteins engaged in native interactions with RNA, in a proteome-wide manner.
A high-throughput in vivo screening method in the mouse for identifying regulators of metastatic colonization Nat. Protoc. (IF 12.423) Pub Date : 2017-11-02 Anneliese O Speak, Agnieszka Swiatkowska, Natasha A Karp, Mark J Arends, David J Adams, Louise van der Weyden
In this protocol, the authors present an experimental metastasis assay in which cancer cells are injected into the tail vein of a mouse, and the resulting secondary organ colonization is assessed, primarily in the lung, 10 d later.
The assembly and use of continuous flow systems for chemical synthesis Nat. Protoc. (IF 12.423) Pub Date : 2017-10-26 Joshua Britton, Timothy F Jamison
Flow chemistry is an attractive alternative to batch chemistry in cases in which improved safety and reaction efficiency can be achieved. This protocol describes the assembly of a continuous flow apparatus from readily available and affordable parts.
Preparation of glycoconjugates from unprotected carbohydrates for protein-binding studies Nat. Protoc. (IF 12.423) Pub Date : 2017-10-26 Christian T Hjuler, Nicolai N Maolanon, Jørgen Sauer, Jens Stougaard, Mikkel B Thygesen, Knud J Jensen
Conjugation of oligosaccharides to probes and surfaces is useful in the development of biochemical assays to assess carbohydrate–protein interactions. In this protocol, conjugation is enabled by using a bifunctional oligo(ethylene glycol) linker.
Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry Nat. Protoc. (IF 12.423) Pub Date : 2017-10-26 Simone Schopper, Abdullah Kahraman, Pascal Leuenberger, Yuehan Feng, Ilaria Piazza, Oliver Müller, Paul J Boersema, Paola Picotti
Many intra- and extracellular signals induce structural changes in proteins. Schopper et al., describe a limited proteolysis–based mass spectrometry (LiP-MS) approach to characterizing these changes at a proteome-wide scale.
Analysis of human cerebrospinal fluid monoamines and their cofactors by HPLC Nat. Protoc. (IF 12.423) Pub Date : 2017-10-19 Marta Batllori, Marta Molero-Luis, Aida Ormazabal, Mercedes Casado, Cristina Sierra, Angels García-Cazorla, Manju Kurian, Simon Pope, Simon J Heales, Rafael Artuch
The levels of monoamines and their cofactors in cerebrospinal fluid are strong indicators for dopamine and serotonin biosynthesis and turnover. This protocol describes a set of HPLC-based approaches for the quantitative detection of these molecules.
Identification of cross talk between SUMOylation and ubiquitylation using a sequential peptide immunopurification approach Nat. Protoc. (IF 12.423) Pub Date : 2017-10-19 Francis P McManus, Frédéric Lamoliatte, Pierre Thibault
Recent studies have uncovered substantial cross talk between the ubiquitylation and SUMOylation pathways. Using sequential affinity purification and mass spectrometry, this protocol enables the identification of proteins that are modified by both pathways.
Cell-derived matrices for studying cell proliferation and directional migration in a complex 3D microenvironment Nat. Protoc. (IF 12.423) Pub Date : 2017-10-19 Riina Kaukonen, Guillaume Jacquemet, Hellyeh Hamidi, Johanna Ivaska
This protocol describes how to produce cell-derived matrices from fibroblasts. These matrices can be used to provide a 3D scaffold for cell culture and to investigate cell behavior in complex microenvironments.
Facile synthesis of gold nanomaterials with unusual crystal structures Nat. Protoc. (IF 12.423) Pub Date : 2017-10-12 Zhanxi Fan, Xiao Huang, Ye Chen, Wei Huang, Hua Zhang
Crystal-phase-controlled synthesis of noble metal nanomaterials is a promising strategy to tune their physicochemical properties. Gold nanomaterials with unusual crystal structures (e.g., hcp, hcp/fcc and 4H) can be prepared under mild conditions.
Simultaneous measurement of sleep and feeding in individual Drosophila Nat. Protoc. (IF 12.423) Pub Date : 2017-10-12 Keith R Murphy, Jin Hong Park, Robert Huber, William W Ja
This protocol describes the construction and use of the Activity Recording CAFE, an automated image-tracking-based system for the integrated measurement of sleep and feeding in individual Drosophila.
Genome-wide mapping of DNase I hypersensitive sites in rare cell populations using single-cell DNase sequencing Nat. Protoc. (IF 12.423) Pub Date : 2017-10-12 James Cooper, Yi Ding, Jiuzhou Song, Keji Zhao
DNase I hypersensitive sites (DHSs) are regions of accessible chromatin that are indicative of regions involved in the regulation of gene expression. scDNase-seq allows genome-wide detection of DHSs from a low number of cells, including single cells.
Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture Nat. Protoc. (IF 12.423) Pub Date : 2017-10-05 Nikolce Gjorevski, Matthias P Lutolf
This protocol describes the synthesis and application of hydrogel matrices comprising a poly(ethylene glycol) backbone, functionalized with cell adhesion cues and laminin-111. Uses include expanding stem cells and differentiating them into organoids.
A cerebellar window for intravital imaging of normal and disease states in mice Nat. Protoc. (IF 12.423) Pub Date : 2017-10-05 Vasileios Askoxylakis, Mark Badeaux, Sylvie Roberge, Ana Batista, Ned Kirkpatrick, Matija Snuderl, Zohreh Amoozgar, Giorgio Seano, Gino B Ferraro, Sampurna Chatterjee, Lei Xu, Dai Fukumura, Dan G Duda, Rakesh K Jain
In this protocol, the skull overlying the cerebellum is removed and a window is applied, enabling intravital imaging to provide a detailed characterization of dynamic processes in this region of the mouse brain.
Total chemical synthesis of histones and their analogs, assisted by native chemical ligation and palladium complexes Nat. Protoc. (IF 12.423) Pub Date : 2017-10-05 Suman Kumar Maity, Muhammad Jbara, Guy Mann, Guy Kamnesky, Ashraf Brik
Chemical synthesis of proteins (e.g., histones) allows precise insertion of modified amino acids. This protocol uses palladium chemistry to remove protecting groups and a removable solubilizing tag for the synthesis of lipophilic peptide segments.
Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces Nat. Protoc. (IF 12.423) Pub Date : 2017-10-05 Richard Newton, Martin Delguste, Melanie Koehler, Andra C Dumitru, Pawel R Laskowski, Daniel J Müller, David Alsteens
This protocol describes how to combine confocal and atomic force microscopy (AFM) to study the interactions between single viruses and their cell-surface receptors on live cells.
Base-resolution stratification of cancer mutations using functional variomics Nat. Protoc. (IF 12.423) Pub Date : 2017-10-05 Song Yi, Ning-Ning Liu, Limei Hu, Hui Wang, Nidhi Sahni
This massively parallel pipeline enables high-throughput generation and confirmation of variants by Gateway cloning, barcoding, and next-generation sequencing, and their stratification by multiplexed interaction profiling and experimental validation.
Generation and use of a humanized bone-marrow-ossicle niche for hematopoietic xenotransplantation into mice Nat. Protoc. (IF 12.423) Pub Date : 2017-09-21 Andreas Reinisch, David Cruz Hernandez, Katharina Schallmoser, Ravindra Majeti
Humanized bone-marrow-ossicle niches are formed in mice via in situ differentiation of bone-marrow-derived mesenchymal stromal cells and can be used for transplantation of normal and malignant human hematopoietic cells.
Genetically encoded releasable photo-cross-linking strategies for studying protein–protein interactions in living cells Nat. Protoc. (IF 12.423) Pub Date : 2017-09-21 Yi Yang, Haiping Song, Dan He, Shuai Zhang, Shizhong Dai, Xiao Xie, Shixian Lin, Ziyang Hao, Huangtao Zheng, Peng R Chen
This protocol describes strategies for the characterization of transient protein–protein interactions and their interaction interfaces via genetically encoded releasable photo-cross-linkers.
Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity Nat. Protoc. (IF 12.423) Pub Date : 2017-09-21 Marcin Poreba, Guy S Salvesen, Marcin Drag
This protocol describes HyCoSuL, an approach that uses tetrapeptides containing natural and >100 unnatural amino acids to screen for protease substrate specificity and to engineer highly active and selective substrates and activity-based probes.
Lab-scale production of anhydrous diazomethane using membrane separation technology Nat. Protoc. (IF 12.423) Pub Date : 2017-09-14 Doris Dallinger, C Oliver Kappe
Diazomethane is useful for inserting methyl or methylene groups in organic synthesis. Unfortunately, it is explosive. A tube-in-flask reactor where the Teflon AF-2400 tube allows only the diazomethane produced to enter the flask can be used to prepare it safely.
Some contents have been Reproduced by permission of The Royal Society of Chemistry.
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