Discovery of potential causative mutations in human coding and noncoding genome with the interactive software BasePlayer Nat. Protoc. (IF 12.423) Pub Date : 2018-10-15 Riku Katainen, Iikki Donner, Tatiana Cajuso, Eevi Kaasinen, Kimmo Palin, Veli Mäkinen, Lauri A. Aaltonen, Esa Pitkänen
Next-generation sequencing (NGS) is routinely applied in life sciences and clinical practice, but interpretation of the massive quantities of genomic data produced has become a critical challenge. The genome-wide mutation analyses enabled by NGS have had a revolutionary impact in revealing the predisposing and driving DNA alterations behind a multitude of disorders. The workflow to identify causative mutations from NGS data, for example in cancer and rare diseases, commonly involves phases such as quality filtering, case–control comparison, genome annotation, and visual validation, which require multiple processing steps and usage of various tools and scripts. To this end, we have introduced an interactive and user-friendly multi-platform-compatible software, BasePlayer, which allows scientists, regardless of bioinformatics training, to carry out variant analysis in disease genetics settings. A genome-wide scan of regulatory regions for mutation clusters can be carried out with a desktop computer in ~10 min with a dataset of 3 million somatic variants in 200 whole-genome-sequenced (WGS) cancers.
Extraction of highly degraded DNA from ancient bones, teeth and sediments for high-throughput sequencing Nat. Protoc. (IF 12.423) Pub Date : 2018-10-15 Nadin Rohland, Isabelle Glocke, Ayinuer Aximu-Petri, Matthias Meyer
DNA preserved in ancient bones, teeth and sediments is typically highly fragmented and present only in minute amounts. Here, we provide a highly versatile silica-based DNA extraction protocol that enables the retrieval of short (≥35 bp) or even ultrashort (≥25 bp) DNA fragments from such material with minimal carryover of substances that inhibit library preparation for high-throughput sequencing. DNA extraction can be performed with either silica spin columns, which offer the most convenient choice for manual DNA extraction, or silica-coated magnetic particles. The latter allow a substantial cost reduction as well as automation on liquid-handling systems. This protocol update replaces a now-outdated version that was published 11 years ago, before high-throughput sequencing technologies became widely available. It has been thoroughly optimized to provide the highest DNA yields from highly degraded samples, as well as fast and easy handling, requiring not more than ~15 min of hands-on time per sample.
Publisher Correction: Assessing spatial pattern separation in rodents using the object pattern separation task Nat. Protoc. (IF 12.423) Pub Date : 2018-10-10 Nick P. van Goethem, Britt T. J. van Hagen, Jos Prickaerts
Publisher Correction: Assessing spatial pattern separation in rodents using the object pattern separation taskPublisher Correction: Assessing spatial pattern separation in rodents using the object pattern separation task, Published online: 10 October 2018; doi:10.1038/s41596-018-0061-2Publisher Correction: Assessing spatial pattern separation in rodents using the object pattern separation task
In vivo pulse labeling of isochronic cohorts of cells in the central nervous system using FlashTag Nat. Protoc. (IF 12.423) Pub Date : 2018-09-26 Subashika Govindan, Polina Oberst, Denis Jabaudon
The tracing of neuronal cell lineages is critical to our understanding of cellular diversity in the CNS. This protocol describes a fluorescence birth-dating technique to label, track and isolate isochronic cohorts of newborn cells in the CNS in vivo in mouse embryos. Injection of carboxyfluorescein esters (CFSEs) into the cerebral ventricle allows pulse labeling of mitotic (M phase) ventricular zone (VZ) progenitors and their progeny across the CNS, a procedure we termed FlashTag. Specificity for M-phase apical progenitors is a result of the somata of these cells transiently contacting the ventricular wall during this cell-cycle phase, exposing them to CFSE injected into the cerebrospinal fluid. Using this approach, the developmental trajectory of progenitors and their daughter neurons can be tracked. Labeled cells can be imaged ex vivo or in fixed tissue, targeted for electrophysiological experiments or isolated using FACS for cell culture or (single-cell) RNA sequencing. Multiple embryos can be labeled within 30 min. The dye is retained for several weeks, allowing labeled cells to be identified postnatally. This protocol describes the labeling procedure using in utero injection, the isolation of live cells using FACS and the processing of labeled tissue for immunohistochemistry.
Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors Nat. Protoc. (IF 12.423) Pub Date : 2018-09-26 Yejun Zou, Aoxue Wang, Mei Shi, Xianjun Chen, Renmei Liu, Ting Li, Chenxia Zhang, Zhuo Zhang, Linyong Zhu, Zhenyu Ju, Joseph Loscalzo, Yi Yang, Yuzheng Zhao
Cellular oxidation–reduction reactions are mainly regulated by pyridine nucleotides (NADPH/NADP+ and NADH/NAD+), thiols, and reactive oxygen species (ROS) and play central roles in cell metabolism, cellular signaling, and cell-fate decisions. A comprehensive evaluation or multiplex analysis of redox landscapes and dynamics in intact living cells is important for interrogating cell functions in both healthy and disease states; however, until recently, this goal has been limited by the lack of a complete set of redox sensors. We recently reported the development of a series of highly responsive, genetically encoded fluorescent sensors for NADPH that substantially strengthen the existing toolset of genetically encoded sensors for thiols, H2O2, and NADH redox states. By combining sensors with unique spectral properties and specific subcellular targeting domains, our approach allows simultaneous imaging of up to four different sensors. In this protocol, we first describe strategies for multiplex fluorescence imaging of these sensors in single cells; then we demonstrate how to apply these sensors to study changes in redox landscapes during the cell cycle, after macrophage activation, and in living zebrafish. This approach can be adapted to different genetically encoded fluorescent sensors and various analytical platforms such as fluorescence microscopy, high-content imaging systems, flow cytometry, and microplate readers. A typical preparation of cells or zebrafish expressing different sensors takes 2–3 d; microscopy imaging or flow-cytometry analysis can be performed within 5–60 min.
Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry Nat. Protoc. (IF 12.423) Pub Date : 2018-09-26 Guojun Han, Matthew H. Spitzer, Sean C. Bendall, Wendy J. Fantl, Garry P. Nolan
Advances in single-cell mass cytometry have increasingly improved highly multidimensional characterization of immune cell heterogeneity. The immunoassay multiplexing capacity relies on monoclonal antibodies labeled with stable heavy-metal isotopes. To date, a variety of rare-earth elements and noble and post-transition metal isotopes have been used in mass cytometry; nevertheless, the methods used for antibody conjugation differ because of the individual metal coordination chemistries and distinct stabilities of various metal cations. Herein, we provide three optimized protocols for conjugating monoclonal IgG antibodies with 48 high-purity heavy-metal isotopes: (i) 38 isotopes of lanthanides, 2 isotopes of indium, and 1 isotope of yttrium; (ii) 6 isotopes of palladium; and (iii) 1 isotope of bismuth. Bifunctional chelating agents containing coordinative ligands of monomeric DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or polymeric pentetic acid (DTPA) were used to stably sequester isotopic cations in aqueous solutions and were subsequently coupled to IgG antibodies using site-specific biorthogonal reactions. Furthermore, quantification methods based on antibody inherent absorption at 280 nm and on extrinsic absorption at 562 nm after staining with bicinchoninic acid (BCA) are reported to determine metal-isotope-tagged antibodies. In addition, a freeze-drying procedure to prepare palladium isotopic mass tags is described. To demonstrate the utility, experiments using six palladium-tagged CD45 antibodies for barcoding assays of live immune cells in cytometry by time-of-flight (CyTOF) are described. Conjugation of pure isotopes of lanthanides, indium, or yttrium takes ~3.5 h. Conjugation of bismuth takes ~4 h. Preparation of palladium mass tags takes ~8 h. Conjugation of pure isotopes of palladium takes ~2.5 h. Antibody titration takes ~4 h.
Mixed-species RNA-seq for elucidation of non-cell-autonomous control of gene transcription Nat. Protoc. (IF 12.423) Pub Date : 2018-09-24 Jing Qiu, Owen Dando, Paul S. Baxter, Philip Hasel, Samuel Heron, T. Ian Simpson, Giles E. Hardingham
Transcriptomic changes induced in one cell type by another mediate many biological processes in the brain and elsewhere; however, achieving artifact-free physical separation of cell types to study them is challenging and generally allows for analysis of only a single cell type. We describe an approach using a co-culture of distinct cell types from different species that enables physical cell sorting to be replaced by in silico RNA sequencing (RNA-seq) read sorting, which is possible because of evolutionary divergence of messenger RNA (mRNA) sequences. As an exemplary experiment, we describe the co-culture of purified neurons, astrocytes, and microglia from different species (12–14 d). We describe how to use our Python tool, Sargasso, to separate the reads from conventional RNA-seq according to species and to eliminate any artifacts borne of imperfect genome annotation (10 h). We show how this procedure, which requires no special skills beyond those that might normally be expected of wet lab and bioinformatics researchers, enables the simultaneous transcriptomic profiling of different cell types, revealing the distinct influence of microglia on astrocytic and neuronal transcriptomes under inflammatory conditions.
Direct fluorine-18 labeling of heat-sensitive biomolecules for positron emission tomography imaging using the Al18F-RESCA method Nat. Protoc. (IF 12.423) Pub Date : 2018-09-24 Frederik Cleeren, Joan Lecina, Jessica Bridoux, Nick Devoogdt, Térence Tshibangu, Catarina Xavier, Guy Bormans
Positron emission tomography (PET) is a quickly expanding, non-invasive molecular imaging technology, and there is high demand for new specific imaging probes. Herein, we present a generic protocol for direct radiolabeling of heat-sensitive biomolecules with the positron-emitting radioisotope fluorine-18 (18F) using the aluminum fluoride restrained complexing agent (Al18F-RESCA) method. The Al18F-RESCA method combines the chemical advantages of a chelator-based radiolabeling method with the unique physical properties of the radionuclide of choice, fluorine-18. Proteins of interest can be conjugated to RESCA via amine coupling using (±)-H3RESCA-TFP, followed by purification using size-exclusion chromatography (SEC). Next, RESCA-derivatized biomolecules can be labeled in one step, at room temperature (~20 °C) in an aqueous medium with aluminum fluoride (Al18F). Al18F-labeled proteins can be obtained with moderate (12–17 GBq/µmol) to good (80–85 GBq/µmol) apparent molar activity, depending on the starting activity of 18F–. In addition, satisfactory radiochemical yields (35–55%, non–decay corrected) and high radiochemical purity (>98%, using gel filtration or solid-phase purification) are obtained. The mild radiolabeling procedure takes 0.5 h to complete and can be used for direct labeling of vector molecules such as peptides, protein scaffolds, and engineered antibody fragments.
Design and operation of reconfigurable two-dimensional DNA molecular arrays Nat. Protoc. (IF 12.423) Pub Date : 2018-09-24 Dongfang Wang, Jie Song, Pengfei Wang, Victor Pan, Yingwei Zhang, Daxiang Cui, Yonggang Ke
Information relay and cascaded transformation are essential in biology and engineering. Imitation of such complex behaviors via synthetic molecular self-assembly at the nanoscale remains challenging. Here we describe the use of structural DNA nanotechnology to realize prescribed, multistep, long-range information relay and cascaded transformation in rationally designed molecular arrays. The engineered arrays provide a controlled platform for studying complex dynamic behaviors of molecular arrays and have a range of potential applications, such as with reconfigurable metamaterials. A reconfigurable array consists of a prescribed number of interconnected dynamic DNA antijunctions. Each antijunction unit consists of four DNA domains of equal length with four dynamic nicking points, which are capable of switching between two stable conformations through an intermediate open conformation. By interconnecting the small DNA antijunctions, one can build custom two-dimensional (2D) molecular ‘domino’ arrays with arbitrary shapes. More important, the DNA molecular arrays are capable of undergoing programmed, multistep, long-range transformation driven by information relay between neighboring antijunction units. The information relay is initiated by the trigger strands under high temperature or formamide concentration. The array’s dynamic behavior can be regulated by external factors such as its shape and size, points of transformation initiation, and/or any engineered information propagation pathways. This protocol provides detailed strategies for designing DNA molecular arrays, as well as procedures for sample production, purification, reconfiguration, and imaging by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The procedure can be completed in 4–7 d.
Genetic lineage tracing of resident stem cells by DeaLT Nat. Protoc. (IF 12.423) Pub Date : 2018-09-24 Lingjuan He, Yan Li, Xiuzhen Huang, Yi Li, Wenjuan Pu, Xueying Tian, Dongqing Cai, Hefeng Huang, Kathy O. Lui, Bin Zhou
Unraveling the fates of resident stem cells during tissue regeneration is an important objective in clinical and basic research. Genetic lineage tracing based on Cre–loxP recombination provides an effective strategy for inferring cell fate and cell conversion in vivo. However, the determination of the exact fates of resident stem cells or their derivatives in disease states and during tissue regeneration remains controversial in many fields of study, partly because of technical limitations associated with Cre-based lineage tracing, such as, for example, off-target labeling. Recently, we generated a new lineage-tracing platform we named DeaLT (dual-recombinase-activated lineage tracing) that uses the Dre–rox recombination system to enhance the precision of Cre-mediated lineage tracing. Here, we describe as an example a detailed protocol using DeaLT to trace the fate of c-Kit+ cardiac stem cells and their derivatives, in the absence of any interference from nontarget cells such as cardiomyocytes, during organ homeostasis and after tissue injury. This lineage-tracing protocol can also be used to delineate the fate of resident stem cells of other organ systems, and takes ~10 months to complete, from mouse crossing to final tissue analysis.
Synthesis of an ultrasensitive BODIPY-derived fluorescent probe for detecting HOCl in live cells Nat. Protoc. (IF 12.423) Pub Date : 2018-09-24 Hao Zhu, Zhen Zhang, Saran Long, Jianjun Du, Jiangli Fan, Xiaojun Peng
Hypochlorous acid (HOCl) is a critical member of the reactive oxygen species (ROS) produced by immune cells to fight infections. On the other hand, HOCl in homeostasis causes oxidative damage to biomolecules and is linked to many diseases, including inflammatory, neurodegenerative, and cardiovascular diseases. Herein, we detail a procedure for the preparation of a boron-dipyrromethene (BODIPY)-derived fluorescent probe for HOCl (BClO) and its application as an imaging reagent in living cells. BClO is synthesized in one pot through a four-step procedure that is nearly the same as that for conventional BODIPY dye preparation, except for the ratio of starting materials. BClO has an extremely rapid response (saturated within seconds) and is ultrasensitive to HOCl. The detection limit of BClO reaches the subnanomolar range, which is the highest HOCl sensitivity to date. Taking advantage of the ultrasensitive character of BClO, we have previously demonstrated its ability to detect endogenous HOCl generated by macrophages and shown that it can also be used to discriminate cancer cell lines (which show high HOCl production) from non-cancer cell lines (which show low HOCl production). The protocol requires ~2 d for probe synthesis and up to ~18 h for fluorescence imaging and flow cytometry assays.
Use of the iNo score to discriminate normal from altered nucleolar morphology, with applications in basic cell biology and potential in human disease diagnostics Nat. Protoc. (IF 12.423) Pub Date : 2018-09-24 Vassiliki Stamatopoulou, Pascaline Parisot, Christophe De Vleeschouwer, Denis L. J. Lafontaine
Ribosome biogenesis is initiated in the nucleolus, a cell condensate essential to gene expression, whose morphology informs cancer pathologists on the health status of a cell. Here, we describe a protocol for assessing, both qualitatively and quantitatively, the involvement of trans-acting factors in the nucleolar structure. The protocol involves use of siRNAs to deplete cells of factors of interest, fluorescence imaging of nucleoli in an automated high-throughput platform, and use of dedicated software to determine an index of nucleolar disruption, the iNo score. This scoring system is unique in that it integrates the five most discriminant shape and textural features of the nucleolus into a parametric equation. Determining the iNo score enables both qualitative and quantitative factor classification with prediction of function (functional clustering), which to our knowledge is not achieved by competing approaches, as well as stratification of their effect (severity of defects) on nucleolar structure. The iNo score has the potential to be useful in basic cell biology (nucleolar structure–function relationships, mitosis, and senescence), developmental and/or organismal biology (aging), and clinical practice (cancer, viral infection, and reproduction). The entire protocol can be completed within 1 week.
Small-seq for single-cell small-RNA sequencing Nat. Protoc. (IF 12.423) Pub Date : 2018-09-24 Michael Hagemann-Jensen, Ilgar Abdullayev, Rickard Sandberg, Omid R Faridani
Small RNAs participate in several cellular processes, including splicing, RNA modification, mRNA degradation, and translational arrest. Traditional methods for sequencing small RNAs require a large amount of cell material, limiting the possibilities for single-cell analyses. We describe Small-seq, a ligation-based method that enables the capture, sequencing, and molecular counting of small RNAs from individual mammalian cells. Here, we provide a detailed protocol for this approach that relies on standard reagents and instruments. The standard protocol captures a complex set of small RNAs, including microRNAs (miRNAs), fragments of tRNAs and small nucleolar RNAs (snoRNAs); however, miRNAs can be enriched through the addition of a size-selection step. Ready-to-sequence libraries can be generated in 2–3 d, starting from cell collection, with additional days needed to computationally map the sequence reads and calculate molecular counts.
Acute and rapid degradation of endogenous proteins by Trim-Away Nat. Protoc. (IF 12.423) Pub Date : 2018-09-24 Dean Clift, Chun So, William A. McEwan, Leo C. James, Melina Schuh
Protein depletion is a key approach to understanding the functions of a protein in a biological system. We recently developed the Trim-Away approach in order to rapidly degrade endogenous proteins without prior modification. Trim-Away is based on the ubiquitin ligase and Fc receptor TRIM21, which recognizes antibody-bound proteins and targets them for degradation by the proteasome. In a typical Trim-Away experiment, protein degradation is achieved in three steps: first, introduction of an antibody against the target protein; second, recruitment of endogenous or exogenous/overexpressed TRIM21 to the antibody–bound target protein; and third, proteasome-mediated degradation of the target protein, antibody and TRIM21 complex. Protein degradation by Trim-Away is acute and rapid, with half-lives of ~10–20 min. The major advantages of Trim-Away over other protein degradation methods are that it can be applied to any endogenous protein without prior modification; that it uses conventional antibodies that are widely available; and that it can be applied to a wide range of cell types, including nondividing primary human cells, for which other loss-of-function assays are challenging. In this protocol, we describe the detailed procedures for antibody preparation and delivery in mouse oocytes and cultured cells via microinjection and electroporation. In addition, we provide recommendations for antibody selection and validation, and for the generation of TRIM21-overexpressing cell lines for cases in which endogenous TRIM21 is limited. A typical Trim-Away experiment takes just a few hours.
Mapping heterogeneity of cellular mechanics by multi-harmonic atomic force microscopy Nat. Protoc. (IF 12.423) Pub Date : 2018-09-14 Yuri M. Efremov, Alexander X. Cartagena-Rivera, Ahmad I. M. Athamneh, Daniel M. Suter, Arvind Raman
The goal of mechanobiology is to understand the links between changes in the physical properties of living cells and normal physiology and disease. This requires mechanical measurements that have appropriate spatial and temporal resolution within a single cell. Conventional atomic force microscopy (AFM) methods that acquire force curves pointwise are used to map the heterogeneous mechanical properties of cells. However, the resulting map acquisition time is much longer than that required to study many dynamic cellular processes. Dynamic AFM (dAFM) methods using resonant microcantilevers are compatible with higher-speed, high-resolution scanning; however, they do not directly acquire force curves and they require the conversion of a limited number of instrument observables to local mechanical property maps. We have recently developed a technique that allows commercial AFM systems equipped with direct cantilever excitation to quantitatively map the viscoelastic properties of live cells. The properties can be obtained at several widely spaced frequencies with nanometer–range spatial resolution and with fast image acquisition times (tens of seconds). Here, we describe detailed procedures for quantitative mapping, including sample preparation, AFM calibration, and data analysis. The protocol can be applied to different biological samples, including cells and viruses. The transition from dAFM imaging to quantitative mapping should be easily achievable for experienced AFM users, who will be able to set up the protocol in <30 min.
Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system Nat. Protoc. (IF 12.423) Pub Date : 2018-09-14 Evelina Tutucci, Maria Vera, Robert H. Singer
The MS2 system has been widely used, in organisms ranging from bacteria to higher eukaryotes, to image single mRNAs in intact cells with high precision. We have recently re-engineered the MS2 system for accurate detection of mRNAs in living Saccharomyces cerevisiae. Previous MS2 systems affected the degradation of the tagged mRNA, which led to accumulation of MS2 fragments and to erroneous conclusions about mRNA localization and expression. Here we describe a step-by-step protocol for the use of our latest MS2 system (MBSV6) for detecting endogenously tagged mRNAs using wide-field fluorescent microscopy in living yeast. The procedure is divided into three stages: tagging of endogenous gene with MBSV6 (~2 weeks), a two-color single-molecule RNA fluorescent in situ hybridization (smFISH) procedure to quantitatively assess whether mRNAs tagged with MS2 and MS2-coat protein (MCP) behave like untagged mRNAs (2 d, plus additional time for quantification), and a procedure to quantify single mRNAs by live imaging using wide-field microscopy (1 d, plus additional time for quantification). With this method it is now possible to interrogate all phases of mRNA expression, from transcription through decay. The described protocol is designed for S. cerevisiae; however, we think that our approach and the considerations discussed here can be extended to Escherichia coli, Drosophila, Caenorhabditis elegans, and mammalian cells.
An experimental murine model to study periodontitis Nat. Protoc. (IF 12.423) Pub Date : 2018-09-14 Julie Marchesan, Mustafa S. Girnary, Li Jing, Michael Zhe Miao, Shaoping Zhang, Lu Sun, Thiago Morelli, Mark H. Schoenfisch, Naohiro Inohara, Steven Offenbacher, Yizu Jiao
Periodontal disease (PD) is a common dental disease associated with the interaction between dysbiotic oral microbiota and host immunity. It is a prevalent disease, resulting in loss of gingival tissue, periodontal ligament, cementum and alveolar bone. PD is a major form of tooth loss in the adult population. Experimental animal models have enabled the study of PD pathogenesis and are used to test new therapeutic approaches for treating the disease. The ligature-induced periodontitis model has several advantages as compared with other models, including rapid disease induction, predictable bone loss and the capacity to study periodontal tissue and alveolar bone regeneration because the model is established within the periodontal apparatus. Although mice are the most convenient and versatile animal models used in research, ligature-induced periodontitis has been more frequently used in large animals. This is mostly due to the technical challenges involved in consistently placing ligatures around murine teeth. To reduce the technical challenge associated with the traditional ligature model, we previously developed a simplified method to easily install a bacterially retentive ligature between two molars for inducing periodontitis. In this protocol, we provide detailed instructions for placement of the ligature and demonstrate how the model can be used to evaluate gingival tissue inflammation and alveolar bone loss over a period of 18 d after ligature placement. This model can also be used on germ-free mice to investigate the role of human oral bacteria in periodontitis in vivo. In conclusion, this protocol enables the mechanistic study of the pathogenesis of periodontitis in vivo.
Generation and assembly of human brain region–specific three-dimensional cultures Nat. Protoc. (IF 12.423) Pub Date : 2018-09-10 Steven A. Sloan, Jimena Andersen, Anca M. Pașca, Fikri Birey, Sergiu P. Pașca
The ability to generate region-specific three-dimensional (3D) models to study human brain development offers great promise for understanding the nervous system in both healthy individuals and patients. In this protocol, we describe how to generate and assemble subdomain-specific forebrain spheroids, also known as brain region–specific organoids, from human pluripotent stem cells (hPSCs). We describe how to pattern the neural spheroids toward either a dorsal forebrain or a ventral forebrain fate, establishing human cortical spheroids (hCSs) and human subpallial spheroids (hSSs), respectively. We also describe how to combine the neural spheroids in vitro to assemble forebrain assembloids that recapitulate the interactions of glutamatergic and GABAergic neurons seen in vivo. Astrocytes are also present in the human forebrain–specific spheroids, and these undergo maturation when the forebrain spheroids are cultured long term. The initial generation of neural spheroids from hPSCs occurs in <1 week, with regional patterning occurring over the subsequent 5 weeks. After the maturation stage, brain region–specific spheroids are amenable to a variety of assays, including live-cell imaging, calcium dynamics, electrophysiology, cell purification, single-cell transcriptomics, and immunohistochemistry studies. Once generated, forebrain spheroids can also be matured for >24 months in culture.
Configuration of electrical spinal cord stimulation through real-time processing of gait kinematics Nat. Protoc. (IF 12.423) Pub Date : 2018-09-06 Marco Capogrosso, Fabien B. Wagner, Jerome Gandar, Eduardo Martin Moraud, Nikolaus Wenger, Tomislav Milekovic, Polina Shkorbatova, Natalia Pavlova, Pavel Musienko, Erwan Bezard, Jocelyne Bloch, Grégoire Courtine
Epidural electrical stimulation (EES) of the spinal cord and real-time processing of gait kinematics are powerful methods for the study of locomotion and the improvement of motor control after injury or in neurological disorders. Here, we describe equipment and surgical procedures that can be used to acquire chronic electromyographic (EMG) recordings from leg muscles and to implant targeted spinal cord stimulation systems that remain stable up to several months after implantation in rats and nonhuman primates. We also detail how to exploit these implants to configure electrical spinal cord stimulation policies that allow control over the degree of extension and flexion of each leg during locomotion. This protocol uses real-time processing of gait kinematics and locomotor performance, and can be configured within a few days. Once configured, stimulation bursts are delivered over specific spinal cord locations with precise timing that reproduces the natural spatiotemporal activation of motoneurons during locomotion. These protocols can also be easily adapted for the safe implantation of systems in the vicinity of the spinal cord and to conduct experiments involving real-time movement feedback and closed-loop controllers.
Fit-free analysis of fluorescence lifetime imaging data using the phasor approach Nat. Protoc. (IF 12.423) Pub Date : 2018-09-06 Suman Ranjit, Leonel Malacrida, David M. Jameson, Enrico Gratton
Fluorescence lifetime imaging microscopy (FLIM) is used in diverse disciplines, including biology, chemistry and biophysics, but its use has been limited by the complexity of the data analysis. The phasor approach to FLIM has the potential to markedly reduce this complexity and at the same time provide a powerful visualization of the data content. Phasor plots for fluorescence lifetime analysis were originally developed as a graphical representation of excited-state fluorescence lifetimes for in vitro systems. The method's simple mathematics and specific rules avoid errors and confusion common in the study of complex and heterogeneous fluorescence. In the case of FLIM, the phasor approach has become a powerful method for simple and fit-free analyses of the information contained in the many thousands of pixels constituting an image. At present, the phasor plot is used not only for FLIM, but also for hyperspectral imaging, wherein phasors provide an unprecedented understanding of heterogeneous fluorescence. Undoubtedly, phasor plots will be increasingly important in the future analysis and understanding of FLIM and hyperspectral confocal imaging. This protocol presents the principle of the method and guides users through one of the popular interfaces for FLIM phasor analysis, namely, the SimFCS software. Implementation of the analysis takes only minutes to complete for a dataset containing hundreds of files.
High-throughput and high-sensitivity phosphoproteomics with the EasyPhos platform Nat. Protoc. (IF 12.423) Pub Date : 2018-09-06 Sean J. Humphrey, Ozge Karayel, David E. James, Matthias Mann
Mass spectrometry has transformed the field of cell signaling by enabling global studies of dynamic protein phosphorylation (‘phosphoproteomics’). Recent developments are enabling increasingly sophisticated phosphoproteomics studies, but practical challenges remain. The EasyPhos workflow addresses these and is sufficiently streamlined to enable the analysis of hundreds of phosphoproteomes at a depth of >10,000 quantified phosphorylation sites. Here we present a detailed and updated workflow that further ensures high performance in sample-limited conditions while also reducing sample preparation time. By eliminating protein precipitation steps and performing the entire protocol, including digestion, in a single 96-well plate, we now greatly minimize opportunities for sample loss and variability. This results in very high reproducibility and a small sample size requirement (≤200 μg of protein starting material). After cell culture or tissue collection, the protocol takes 1 d, whereas mass spectrometry measurements require ~1 h per sample. Applied to glioblastoma cells acutely treated with epidermal growth factor (EGF), EasyPhos quantified 20,132 distinct phosphopeptides from 200 μg of protein in less than 1 d of measurement time, revealing thousands of EGF-regulated phosphorylation events.
Preparation of asymmetric phospholipid vesicles for use as cell membrane models Nat. Protoc. (IF 12.423) Pub Date : 2018-09-06 Milka Doktorova, Frederick A. Heberle, Barbara Eicher, Robert F. Standaert, John Katsaras, Erwin London, Georg Pabst, Drew Marquardt
Freely suspended liposomes are widely used as model membranes for studying lipid–lipid and protein–lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-β-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet’s lipid composition: the overall lipid composition is determined by gas chromatography–mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr3+. The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies.
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).
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