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Current Protocols in Molecular Biology

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https://www.readbyqxmd.com/read/28967997/a-detailed-protocol-for-subcellular-rna-sequencing-subrna-seq
#1
Andreas Mayer, L Stirling Churchman
In eukaryotic cells, RNAs at various maturation and processing levels are distributed across cellular compartments. The standard approach to determine transcript abundance and identity in vivo is RNA sequencing (RNA-seq). RNA-seq relies on RNA isolation from whole-cell lysates and thus mainly captures fully processed, stable, and more abundant cytoplasmic RNAs over nascent, unstable, and nuclear RNAs. Here, we provide a step-by-step protocol for subcellular RNA-seq (subRNA-seq). subRNA-seq allows the quantitative measurement of RNA polymerase II-generated RNAs from the chromatin, nucleoplasm, and cytoplasm of mammalian cells...
October 2, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28967996/single-assay-profiling-of-nucleosome-occupancy-and-chromatin-accessibility
#2
April Cook, Jakub Mieczkowski, Michael Y Tolstorukov
This unit describes a method for determining the accessibility of chromatinized DNA and nucleosome occupancy in the same assay. Enzymatic digestion of chromatin using micrococcal nuclease (MNase) is optimized for liberation, retrieval, and characterization of DNA fragments from chromatin. MNase digestion is performed in a titration series, and the DNA fragments are isolated and sequenced for each individual digest independently. These sequenced fragments are then collectively analyzed in a novel bioinformatics pipeline to produce a metric describing MNase accessibility of chromatin (MACC) and nucleosome occupancy...
October 2, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28967995/casaav-a-crispr-based-platform-for-rapid-dissection-of-gene-function-in-vivo
#3
Nathan J VanDusen, Yuxuan Guo, Weiliang Gu, William T Pu
In vivo loss-of-function studies are currently limited by the need for appropriate conditional knockout alleles. CRISPR/Cas9 is a powerful tool commonly used to induce loss-of-function mutations in vitro. However, CRISPR components have been difficult to deploy in vivo. To address this problem, we developed the CASAAV (CRISPR/Cas9/AAV-based somatic mutagenesis) platform, in which recombinant adeno-associated virus (AAV) is used to deliver tandem guide RNAs and Cre recombinase to Cre-dependent Cas9-P2A-GFP mice...
October 2, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28967994/transposon-insertion-site-sequencing-tis-seq-an-efficient-and-high-throughput-method-for-determining-transposon-insertion-site-s-and-their-relative-abundances-in-a-piggybac-transposon-mutant-pool-by-next-generation-sequencing
#4
Yaligara Veeranagouda, Michel Didier
The PiggyBac (PB) transposon has emerged as a novel mutagenesis tool for understanding gene function and for phenotypic screening in eukaryotes. Successful screening of PB transposon mutants relies on efficient identification of transposon insertion site(s) (TIS) in mutant cells. However, currently available methods suffer from time-consuming steps. Here, we present the method for transposon insertion site sequencing (TIS-Seq) for high-throughput identification of TIS in transposon mutants. TIS-Seq provides qualitative and quantitative information on mutants present in a given PB transposon mutant library...
October 2, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28967993/production-of-purified-casrnps-for-efficacious-genome-editing
#5
Emily Lingeman, Chris Jeans, Jacob E Corn
CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators...
October 2, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28678443/mitochondrial-ribosome-mitoribosome-profiling-for-monitoring-mitochondrial-translation-in-vivo
#6
Mary T Couvillion, L Stirling Churchman
Translation in the mitochondria is regulated by mechanisms distinct from those acting in the cytosol and in bacteria, yet precise methods for investigating it have lagged behind. This unit describes an approach, mitochondrial ribosome (mitoribosome) profiling, to quantitatively monitor mitochondrial translation with high temporal and spatial resolution in Saccharomyces cerevisiae. Mitoribosomes are immunoprecipitated from whole-cell lysate and the protected mRNA fragments are isolated. These fragments are then converted to sequencing libraries or analyzed by northern blot hybridization to reveal the distribution of mitoribosomes across the mitochondrial transcriptome...
July 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28678442/crispr-cas9-directed-gene-editing-for-the-generation-of-loss-of-function-mutants-in-high-throughput-zebrafish-f0-screens
#7
Sunita S Shankaran, Timothy J Dahlem, Brent W Bisgrove, H Joseph Yost, Martin Tristani-Firouzi
The ability to perform reverse genetics in the zebrafish model organism has been greatly advanced with the advent of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated) system. The high level of efficiency in generating mutations when using the CRISPR/Cas9 system combined with the rapid generation time of the zebrafish model organism has made the possibility of performing F0 screens in this organism a reality. This unit describes a detailed protocol for performing an F0 screen using the CRISPR/Cas9 system in zebrafish starting with the design and production of custom CRISPR/Cas9 reagents for injection...
July 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28678441/nebnext-direct-a-novel-rapid-hybridization-based-approach-for-the-capture-and-library-conversion-of-genomic-regions-of-interest
#8
Amy B Emerman, Sarah K Bowman, Andrew Barry, Noa Henig, Kruti M Patel, Andrew F Gardner, Cynthia L Hendrickson
Next-generation sequencing (NGS) is a powerful tool for genomic studies, translational research, and clinical diagnostics that enables the detection of single nucleotide polymorphisms, insertions and deletions, copy number variations, and other genetic variations. Target enrichment technologies improve the efficiency of NGS by only sequencing regions of interest, which reduces sequencing costs while increasing coverage of the selected targets. Here we present NEBNext Direct(®) , a hybridization-based, target-enrichment approach that addresses many of the shortcomings of traditional target-enrichment methods...
July 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28678440/automated-computational-processing-of-3-d-mr-images-of-mouse-brain-for-phenotyping-of-living-animals
#9
Christopher S Medina, Brett Manifold-Wheeler, Aaron Gonzales, Elaine L Bearer
Magnetic resonance (MR) imaging provides a method to obtain anatomical information from the brain in vivo that is not typically available by optical imaging because of this organ's opacity. MR is nondestructive and obtains deep tissue contrast with 100-µm(3) voxel resolution or better. Manganese-enhanced MRI (MEMRI) may be used to observe axonal transport and localized neural activity in the living rodent and avian brain. Such enhancement enables researchers to investigate differences in functional circuitry or neuronal activity in images of brains of different animals...
July 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28369679/preparing-viable-single-cells-from-human-tissue-and-tumors-for-cytomic-analysis
#10
Nalin Leelatian, Deon B Doxie, Allison R Greenplate, Justine Sinnaeve, Rebecca A Ihrie, Jonathan M Irish
Mass cytometry is a single-cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >10(4) relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies where investigators used mass cytometry to measure protein and phospho-protein expression in millions of cells, characterize rare cell types in healthy and diseased tissues, and reveal novel, unexpected cells. However, these advances largely occurred in studies of blood, lymphoid tissues, and bone marrow, since the cells in these tissues are readily obtained in single-cell suspensions...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28369678/mapping-transposon-insertions-in-bacterial-genomes-by-arbitrarily-primed-pcr
#11
José T Saavedra, Julia A Schwartzman, Michael S Gilmore
Transposons can be used to easily generate and label the location of mutations throughout bacterial and other genomes. Transposon insertion mutants may be screened for a phenotype as individual isolates, or by selection applied to a pool of thousands of mutants. Identifying the location of a transposon insertion is critical for connecting phenotype to the genetic lesion. In this unit, we present an easy and detailed approach for mapping transposon insertion sites using arbitrarily-primed PCR (AP-PCR). Two rounds of PCR are used to (1) amplify DNA spanning the transposon insertion junction, and (2) increase the specific yield of transposon insertion junction fragments for sequence analysis...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28369677/direct-isolation-of-seamless-mutant-bacterial-artificial-chromosomes
#12
George T Lyozin, Yasuhiro Kosaka, Gourab Bhattacharje, H Joseph Yost, Luca Brunelli
Seamless (i.e., without unwanted DNA sequences) mutant bacterial artificial chromosomes (BACs) generated via recombination-mediated genetic engineering (recombineering) are better suited to study gene function compared to complementary DNA (cDNA) because they contain only the specific mutation and provide all the regulatory sequences required for in vivo gene expression. However, precisely mutated BACs are typically rare (∼1:1,000 to 1:100,000), making their isolation quite challenging. Although these BACs have been classically isolated by linking the mutation to additional genes, i...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28369676/transcriptome-wide-identification-of-rna-binding-protein-binding-sites-using-photoactivatable-ribonucleoside-enhanced-crosslinking-immunoprecipitation-par-clip
#13
Henrike Maatz, Marcin Kolinski, Norbert Hubner, Markus Landthaler
RNA-binding proteins (RBPs) mediate important co- and post-transcriptional gene regulation by binding pre-mRNA in a sequence- and/or structure-specific manner. For a comprehensive understanding of RBP function, transcriptome-wide mapping of the RNA-binding sites is essential, and CLIP-seq methods have been developed to elucidate protein/RNA interactions at high resolution. CLIP-seq combines protein/RNA UV-crosslinking with immunoprecipitation (CLIP) followed by high-throughput sequencing of crosslinked RNA fragments...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28369675/analysis-of-membrane-protein-interactions-with-a-bacterial-adenylate-cyclase-based-two-hybrid-bacth-technique
#14
Scot P Ouellette, Gouzel Karimova, Marilyne Davi, Daniel Ladant
The bacterial two-hybrid (BACTH, for "Bacterial Adenylate Cyclase-based Two-Hybrid") technique is a simple and fast genetic approach to analyze protein-protein interactions in vivo. In this system, the proteins of interest are genetically fused to two complementary fragments from the catalytic domain of Bordetella pertussis adenylate cyclase and co-expressed in strains of Escherichia coli deficient in adenylate cyclase. Association of the hybrid proteins restores synthesis of cyclic AMP (cAMP), which then triggers the expression of catabolic operons such as the lactose operon or the maltose regulon...
April 3, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060411/scarless-cas9-assisted-recombineering-no-scar-in-escherichia-coli-an-easy-to-use-system-for-genome-editing
#15
Christopher R Reisch, Kristala L J Prather
The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair...
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060410/preparation-of-cell-cultures-and-vaccinia-virus-stocks
#16
Catherine A Cotter, Patricia L Earl, Linda S Wyatt, Bernard Moss
The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley & Sons, Inc.
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060409/immunization-of-mice
#17
Tomás Maira-Litrán
The selection of an appropriate immunization strategy depends largely on the properties of an antigen, including its nature, purity, solubility, and availability. This unit describes the critical steps in the production of monoclonal and polyclonal antibodies against soluble molecules (such as proteins, peptides, polysaccharides, oligosaccharides, or hapten-conjugate vaccines), complex antigens (such as whole pathogens or outer membrane vesicles), and antigens embedded in or eluted from gel matrix following electrophoresis...
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060408/next-generation-sequencing-for-identification-of-ems-induced-mutations-in-caenorhabditis-elegans
#18
Nicolas J Lehrbach, Fei Ji, Ruslan Sadreyev
Forward genetic analysis using chemical mutagenesis in model organisms is a powerful tool for investigation of molecular mechanisms in biological systems. In the nematode, Caenorhabditis elegans, mutagenesis screens using ethyl methanesulfonate (EMS) have led to important insights into genetic control of animal development and physiology. A major bottleneck to this approach is identification of the causative mutation underlying a phenotype of interest. In the past, this has required time-consuming genetic mapping experiments...
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060407/mammalian-cell-tissue-culture-techniques
#19
Katy Phelan, Kristin M May
Cultured mammalian cells are used extensively in molecular biology studies. A number of special skills are required in order to preserve the structure, function, behavior, and biology of cells in culture. This appendix describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2017 by John Wiley & Sons, Inc.
January 5, 2017: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28060406/a-simple-protoplast-based-method-for-screening-potent-artificial-mirna-for-maximal-gene-silencing-in-arabidopsis
#20
Nannan Zhang, Dandan Zhang, Jian-Feng Li
The unpredictability of in planta silencing efficiency of artificial micro RNAs (amiRNAs) has greatly limited the application of the amiRNA technology in basic and applied plant research. This unit describes a simple and robust method called the epitope-tagged protein-based amiRNA (ETPamir) screen for identifying the most effective amiRNA for silencing a particular Arabidopsis gene. After selecting three to four amiRNA candidates designed by the WMD3 web server for the target gene, the silencing efficiencies of the amiRNA candidates can be compared by co-expressing individual amiRNAs with the target mRNA encoding an epitope-tagged target protein in Arabidopsis mesophyll protoplasts...
January 5, 2017: Current Protocols in Molecular Biology
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