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Current Protocols in Molecular Biology

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https://www.readbyqxmd.com/read/29851299/using-barcoded-hiv-ensembles-b-hive-for-single-provirus-transcriptomics
#1
Heng-Chang Chen, Eduard Zorita, Guillaume J Filion
The latent HIV reservoir is the main barrier to curing AIDS, because infected cells escape the immune system and antiretroviral therapies. Developing new treatment strategies requires technologies to trace latent proviruses. Here, we describe a genome-wide technique called Barcoded HIV Ensembles (B-HIVE) to measure HIV expression at the single provirus level. The principle of B-HIVE is to tag the genome of HIV with DNA barcodes to trace viral transcripts produced by single proviruses in an infected cell population...
April 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29851291/overview-of-next-generation-sequencing-technologies
#2
Barton E Slatko, Andrew F Gardner, Frederick M Ausubel
High throughput DNA sequencing methodology (next generation sequencing; NGS) has rapidly evolved over the past 15 years and new methods are continually being commercialized. As the technology develops, so do increases in the number of corresponding applications for basic and applied science. The purpose of this review is to provide a compendium of NGS methodologies and associated applications. Each brief discussion is followed by web links to the manufacturer and/or web-based visualizations. Keyword searches, such as with Google, may also provide helpful internet links and information...
April 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29851283/introduction-to-single-cell-rna-sequencing
#3
Thale Kristin Olsen, Ninib Baryawno
During the last decade, high-throughput sequencing methods have revolutionized the entire field of biology. The opportunity to study entire transcriptomes in great detail using RNA sequencing (RNA-seq) has fueled many important discoveries and is now a routine method in biomedical research. However, RNA-seq is typically performed in "bulk," and the data represent an average of gene expression patterns across thousands to millions of cells; this might obscure biologically relevant differences between cells...
April 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29851250/human-induced-pluripotent-stem-cell-production-and-expansion-from-blood-using-a-non-integrating-viral-reprogramming-vector
#4
Arun Sharma, Michael Mücke, Christine E Seidman
We describe a method to transform blood lymphocytes into human-induced pluripotent stem cells by delivering four transcription factors with a non-integrative virus. Using human peripheral blood mononuclear cells (PBMCs) as the source cell type for hiPSC reprogramming is advantageous since blood samples are rapidly and safely obtained from nearly-all subjects. Reprogramming factors needed to make hiPSCs are introduced by infecting the PBMCs with non-integrating Sendai virus vectors. Reprogrammed cells can subsequently be quickly expanded for downstream use...
April 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29851244/the-use-of-the-fluidigm-c1-for-rna-expression-analyses-of-single-cells
#5
Daniel M DeLaughter
Understanding the transcriptional heterogeneity that occurs on the level of a single cell is critical to understanding the gene-regulatory mechanisms underlying development and disease. Population-level whole-transcriptome profiling approaches average gene expression across thousands to millions of cells and are unable to delineate the transcriptional signature of individual cells. Considerable biological heterogeneity between individual cells arises from differences in cell lineage, environment, or response to stimulus...
April 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337376/crispr-cas9-mediated-genome-editing-in-epstein-barr-virus-transformed-lymphoblastoid-b-cell-lines
#6
Sizun Jiang, Liang Wei Wang, Michael J Walsh, Stephen J Trudeau, Catherine Gerdt, Bo Zhao, Benjamin E Gewurz
Epstein-Barr virus (EBV) efficiently transforms primary human B cells into immortalized lymphoblastoid cell lines (LCLs), which are extensively used in human genetic, immunological and virological studies. LCLs provide unlimited sources of DNA for genetic investigation, but can be difficult to manipulate, for instance because low retroviral or lentiviral transduction frequencies hinder experiments that require co-expression of multiple components. This unit details Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 engineering for robust LCL genome editing...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337375/transfection-by-electroporation
#7
Huntington Potter, Richard Heller
Electroporation-the use of high-voltage electric shocks to introduce DNA into cells-can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques. This unit describes electroporation of mammalian cells, including ES cells for the preparation of knock-out, knock-in, and transgenic mice. Protocols are described for the use of electroporation in vivo to perform gene therapy for cancer therapy and DNA vaccination...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337374/pooled-lentiviral-delivery-genetic-screens
#8
Federica Piccioni, Scott T Younger, David E Root
Pooled cell-based screens of mammalian genetic perturbations enable systematic large-scale, even genome-scale, evaluation of gene function. Pooled screens introduce genetic perturbations into a cell population through viral transduction such that each cell integrates into its DNA a single or small number of library perturbations with barcodes identifying the perturbations. One then selects and physically isolates the subset of cells that exhibit the phenotype of interest. Sequencing the barcodes in the hit cells reveals which genes favored or inhibited the hit phenotype...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337373/making-use-of-cancer-genomic-databases
#9
Chad J Creighton
The vast amounts of genomic data now deposited in public repositories represent rich resources for cancer researchers. Large-scale genomics initiatives such as The Cancer Genome Atlas have made available data from multiple molecular profiling platforms (e.g., somatic mutation, RNA and protein expression, and DNA methylation) for the same set of over 10,000 human tumors. There has been much collective effort toward providing user-friendly software tools for biologists lacking computational skills to ask questions of large-scale genomic datasets...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337372/crispr-cas9-edited-site-sequencing-cres-seq-an-efficient-and-high-throughput-method-for-the-selection-of-crispr-cas9-edited-clones
#10
Yaligara Veeranagouda, Delphine Debono-Lagneaux, Hamida Fournet, Gilbert Thill, Michel Didier
The emergence of clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) gene editing systems has enabled the creation of specific mutants at low cost, in a short time and with high efficiency, in eukaryotic cells. Since a CRISPR-Cas9 system typically creates an array of mutations in targeted sites, a successful gene editing project requires careful selection of edited clones. This process can be very challenging, especially when working with multiallelic genes and/or polyploid cells (such as cancer and plants cells)...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337371/avian-retrovirus-mediated-tumor-specific-gene-knockout
#11
Wei Wang, Bingning Dong, Feng Yang
The RCAS (replication-competent avian sarcoma leukosis virus long-terminal repeat with splice acceptor)-TVA (tumor virus A) gene delivery system has been successfully used in modeling human cancers. Based on this, we have recently developed a novel RCI-Oncogene (RCAS-Cre-IRES-Oncogene) gene delivery system that can be used to efficiently manipulate gene expression in spontaneous tumors in vivo. We used this system for tumor gene knockout (TuKO) and demonstrated a crucial role of FGFR1 in driving mammary tumor metastasis...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/29337370/modulating-gene-expression-in-epstein-barr-virus-ebv-positive-b-cell-lines-with-crispra-and-crispri
#12
Liang Wei Wang, Stephen J Trudeau, Chong Wang, Catherine Gerdt, Sizun Jiang, Bo Zhao, Benjamin E Gewurz
Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus...
January 16, 2018: Current Protocols in Molecular Biology
https://www.readbyqxmd.com/read/28967997/a-detailed-protocol-for-subcellular-rna-sequencing-subrna-seq
#13
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
#14
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
#15
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
#16
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
#17
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
#18
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-f-0-screens
#19
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
#20
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
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