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Nature Protocols

Amy Reilein, Elisa Cimetta, Nina M Tandon, Daniel Kalderon, Gordana Vunjak-Novakovic
The version of this paper originally published contained an incorrect unit abbreviation in Step 21: "0.20 g/mL" should have been "0.20 mg/mL." In addition, the first sentence in Step 33 should have read "Use a second pipette with a cut-off pipette tip to add Matrigel to the center well," instead of "Use a second pipette to cut off the tip of the pipette and add Matrigel to the center well." These errors have been corrected in the PDF and HTML versions of the protocol.
February 15, 2019: Nature Protocols
Zhengxiao Fan, Hong Zhu, Tingting Zhou, Sheng Wang, Yan Wu, Hailan Hu
Investigation of the neural mechanisms underlying social hierarchy requires a reliable and effective behavioral test. The tube test is a simple and robust behavioral assay that we recently validated as a reliable measure of social hierarchy in mice. The test was demonstrated to produce results largely consistent with the results seen when using other dominance measures, including the warm spot test, territory urine marking or the courtship ultrasound vocalization test. Here, we describe a step-by-step procedure to use the tube test to measure dominance within a cage of four male C57/BL6 mice as an example application...
February 15, 2019: Nature Protocols
Rebecca E McKenzie, Cristóbal Almendros, Jochem N A Vink, Stan J J Brouns
CRISPR-Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 105 cells...
February 11, 2019: Nature Protocols
Viola Halder, Caroline B M Porter, Alejandro Chavez, Rebecca S Shapiro
The study of fungal pathogens is of immediate importance, yet progress is hindered by the technical challenges of genetic manipulation. For Candida species, their inability to maintain plasmids, unusual codon usage, and inefficient homologous recombination are among the obstacles limiting efficient genetic manipulation. New advances in genomic biotechnologies-particularly CRISPR-based tools-have revolutionized genome editing for many fungal species. Here, we present a protocol for CRISPR-Cas9-based manipulation in Candida albicans using a modified gene-drive-based strategy that takes ~1 month to complete...
February 8, 2019: Nature Protocols
Andrew D Duckworth, Pier Federico Gherardini, Martina Sykorova, Faten Yasin, Garry P Nolan, Joseph R Slupsky, Nagesh Kalakonda
Advances in single-cell analysis technologies are providing novel insights into phenotypic and functional heterogeneity within seemingly identical cell populations. RNA within single cells can be analyzed using unbiased sequencing protocols or through more targeted approaches using in situ hybridization (ISH). The proximity ligation assay for RNA (PLAYR) approach is a sensitive and high-throughput technique that relies on in situ and proximal ligation to measure at least 27 specific RNAs by flow or mass cytometry...
February 6, 2019: Nature Protocols
Binbin Wang, Mei Wang, Wubing Zhang, Tengfei Xiao, Chen-Hao Chen, Alexander Wu, Feizhen Wu, Nicole Traugh, Xiaoqing Wang, Ziyi Li, Shenglin Mei, Yingbo Cui, Sailing Shi, Jesse Jonathan Lipp, Matthias Hinterndorfer, Johannes Zuber, Myles Brown, Wei Li, X Shirley Liu
Genome-wide screening using CRISPR coupled with nuclease Cas9 (CRISPR-Cas9) is a powerful technology for the systematic evaluation of gene function. Statistically principled analysis is needed for the accurate identification of gene hits and associated pathways. Here, we describe how to perform computational analysis of CRISPR screens using the MAGeCKFlute pipeline. MAGeCKFlute combines the MAGeCK and MAGeCK-VISPR algorithms and incorporates additional downstream analysis functionalities. MAGeCKFlute is distinguished from other currently available tools by its comprehensive pipeline, which contains a series of functions for analyzing CRISPR screen data...
February 1, 2019: Nature Protocols
Jie Lin, Yujian Wen, Sha He, Xiaoxue Yang, Hai Zhang, Hao Zhu
Abundant long, noncoding RNAs (lncRNAs) in mammals can bind to DNA sequences and recruit histone- and DNA-modifying enzymes to binding sites to epigenetically regulate target genes. However, most lncRNAs' binding motifs and target sites are unknown. The large numbers of lncRNAs and target sites in the whole genome make it infeasible to examine lncRNA binding to DNA purely experimentally. Here, we report a protocol for lncRNA/DNA-binding analysis that is built upon a database containing the GENCODE-annotated human and mouse lncRNAs, the orthologs of these lncRNAs in 17 mammals, and the genome sequences of the 17 mammals...
January 30, 2019: Nature Protocols
Ji-Feng Fei, Wilson Pak-Kin Lou, Dunja Knapp, Prayag Murawala, Tobias Gerber, Yuka Taniguchi, Sergej Nowoshilow, Shahryar Khattak, Elly M Tanaka
In the version of this protocol originally published, the recipe for CAS9 buffer was incorrectly identified as a recipe for sodium acetate solution, and vice versa. These errors have been corrected in the PDF and HTML versions of the paper.
January 29, 2019: Nature Protocols
Susan E Henkelis, Michal Mazur, Cameron M Rice, Giulia P M Bignami, Paul S Wheatley, Sharon E Ashbrook, Jiří Čejka, Russell E Morris
High-silica zeolites, some of the most important and widely used catalysts in industry, have potential for application across a wide range of traditional and emerging technologies. The many structural topologies of zeolites have a variety of potential uses, so a strong drive to create new zeolites exists. Here, we present a protocol, the assembly-disassembly-organization-reassembly (ADOR) process, for a relatively new method of preparing these important solids. It allows the synthesis of new high-silica zeolites (Si/Al >1,000), whose synthesis is considered infeasible with traditional (solvothermal) methods, offering new topologies that may find novel applications...
January 25, 2019: Nature Protocols
Guadalupe Lorenzatti Hiles, Angelica L Cates, Layla El-Sawy, Kathleen C Day, Luke J Broses, Amy L Han, Hannah L Briggs, Amir Emamdjomeh, Andrew Chou, Ethan V Abel, Monica Liebert, Phillip L Palmbos, Aaron M Udager, Evan T Keller, Mark L Day
The invasion of bladder cancer into the sub-urothelial muscle and vasculature are key determinants leading to lethal metastatic progression. However, the molecular basis is poorly understood, partly because of the lack of uncomplicated and reliable models that recapitulate the biology of locally invasive disease. We developed a surgical grafting technique, characterized by a simple, rapid, reproducible and high-efficiency approach, to recapitulate the pathobiological events of human bladder cancer invasion in mice...
January 25, 2019: Nature Protocols
Alyssa J Miller, Briana R Dye, Daysha Ferrer-Torres, David R Hill, Arend W Overeem, Lonnie D Shea, Jason R Spence
The lung epithelium is derived from the endodermal germ layer, which undergoes a complex series of endoderm-mesoderm-mediated signaling events to generate the final arborized network of conducting airways (bronchi, bronchioles) and gas-exchanging units (alveoli). These stages include endoderm induction, anterior-posterior and dorsal-ventral patterning, lung specification, lung budding, branching morphogenesis, and, finally, maturation. Here we describe a protocol that recapitulates several of these milestones in order to differentiate human pluripotent stem cells (hPSCs) into ventral-anterior foregut spheroids and further into two distinct types of organoids: human lung organoids and bud tip progenitor organoids...
January 21, 2019: Nature Protocols
Jüri Reimand, Ruth Isserlin, Veronique Voisin, Mike Kucera, Christian Tannus-Lopes, Asha Rostamianfar, Lina Wadi, Mona Meyer, Jeff Wong, Changjiang Xu, Daniele Merico, Gary D Bader
Pathway enrichment analysis helps researchers gain mechanistic insight into gene lists generated from genome-scale (omics) experiments. This method identifies biological pathways that are enriched in a gene list more than would be expected by chance. We explain the procedures of pathway enrichment analysis and present a practical step-by-step guide to help interpret gene lists resulting from RNA-seq and genome-sequencing experiments. The protocol comprises three major steps: definition of a gene list from omics data, determination of statistically enriched pathways, and visualization and interpretation of the results...
January 21, 2019: Nature Protocols
Scott W Simpkins, Raamesh Deshpande, Justin Nelson, Sheena C Li, Jeff S Piotrowski, Henry Neil Ward, Yoko Yashiroda, Hiroyuki Osada, Minoru Yoshida, Charles Boone, Chad L Myers
The construction of genome-wide mutant collections has enabled high-throughput, high-dimensional quantitative characterization of gene and chemical function, particularly via genetic and chemical-genetic interaction experiments. As the throughput of such experiments increases with improvements in sequencing technology and sample multiplexing, appropriate tools must be developed to handle the large volume of data produced. Here, we describe how to apply our approach to high-throughput, fitness-based profiling of pooled mutant yeast collections using the BEAN-counter software pipeline (Barcoded Experiment Analysis for Next-generation sequencing) for analysis...
January 11, 2019: Nature Protocols
Rosemary C Challis, Sripriya Ravindra Kumar, Ken Y Chan, Collin Challis, Keith Beadle, Min J Jang, Hyun Min Kim, Pradeep S Rajendran, John D Tompkins, Kalyanam Shivkumar, Benjamin E Deverman, Viviana Gradinaru
We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression...
January 9, 2019: Nature Protocols
Jian Yang, David J Ryan, Guocheng Lan, Xiangang Zou, Pentao Liu
Molecular and embryology studies have demonstrated that mouse pre-implantation embryo development is a process of progressive cell fate determination. At the time of implantation, three cell lineages are present in the developing blastocyst: the trophectoderm (TE), the epiblast (Epi) and the primitive endoderm (PrE). From these early embryo cells, trophoblast stem (TS) cells, embryonic stem (ES) cells and extra-embryonic endoderm stem (XEN) cells can be derived. Recently, we derived stem cells with blastomere-like features from mouse cleavage-stage embryos, which we named expanded-potential stem cells (EPSCs)...
January 7, 2019: Nature Protocols
Jie Chen, Lukas D Landegger, Yao Sun, Jun Ren, Nir Maimon, Limeng Wu, Mei R Ng, John W Chen, Na Zhang, Yingchao Zhao, Xing Gao, Takeshi Fujita, Sylvie Roberge, Peigen Huang, Rakesh K Jain, Scott R Plotkin, Konstantina M Stankovic, Lei Xu
Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is the lack of animal models that allow the full spectrum of research into the biology and molecular mechanisms of NF2 tumor progression, as well as the effects on neurological function. In this protocol, we describe how to inject schwannoma cells into the mouse brain cerebellopontine angle (CPA) region...
January 7, 2019: Nature Protocols
Kevin S Chen, Lisa M McGinley, Osama N Kashlan, John M Hayes, Elizabeth S Bruno, Josh S Chang, Faye E Mendelson, Maegan A Tabbey, Karl Johe, Stacey A Sakowski, Eva L Feldman
Despite decades of research, pharmacological therapies for spinal cord motor pathologies are limited. Alternatives using macromolecular, viral, or cell-based therapies show early promise. However, introducing these substances into the spinal cord, past the blood-brain barrier, without causing injury is challenging. We describe a technique for intraspinal injection targeting the lumbar ventral horn in rodents. This technique preserves motor performance and has a proven track record of translation into phase 1 and 2 clinical trials in amyotrophic lateral sclerosis (ALS) patients...
January 4, 2019: Nature Protocols
Flavio C Lombardo, Valérian Pasche, Gordana Panic, Yvette Endriss, Jennifer Keiser
Drug discovery for schistosomiasis is still limited to a handful of academic laboratories worldwide, with only a few novel antischistosomal lead compounds being actively researched. Despite recent international mobilization against the disease to stimulate and promote antischistosomal drug discovery, setting up a drug-screening flow with schistosome parasites remains challenging. Whereas numerous different protocols to obtain and cultivate schistosomes have been published, those describing the drug-screening process are scarce, and none gather together parasite cultivation and early drug discovery procedures...
January 4, 2019: Nature Protocols
Beatriz Alvarez-Castelao, Christoph T Schanzenbächer, Julian D Langer, Erin M Schuman
A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L274G), which enables the labeling of nascent proteins with the non-canonical amino acid azidonorleucine (ANL). This amino acid can be conjugated to different affinity tags by click chemistry. After affinity purification (AP), the labeled proteins can be identified by tandem mass spectrometry (MS/MS)...
January 4, 2019: Nature Protocols
Lorena Hidalgo San Jose, Robert A J Signer
Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and homeostasis. Most methods used to measure protein synthesis depend on metabolically labeling large numbers of cells with radiolabeled amino acids or amino acid analogs. Because these methods typically depend on specialized growth conditions, they have been largely restricted to yeast, bacteria and cell lines. Application of these techniques to investigating protein synthesis within mammalian systems in vivo has been challenging...
January 4, 2019: Nature Protocols
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