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Latest Articles

Analysis of Cellular DNA Content by Flow Cytometry
Zbigniew Darzynkiewicz, Xuan Huang, Hong Zhao
Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this
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Click Chemistry for Analysis of Cell Proliferation in Flow Cytometry
Scott T. Clarke, Veronica Calderon, Jolene A. Bradford
The measurement of cellular proliferation is fundamental to the assessment of cellular health, genotoxicity, and the evaluation of drug efficacy. Labeling, detection, and quantification of cells in the synthesis phase of cell cycle progression are not only important for characterizing basic biology,
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CyTOF Measurement of Immunocompetence Across Major Immune Cell Types
Priyanka B. Subrahmanyam, Holden T. Maecker
The central role of the immune system is becoming appreciated in a wide variety of diseases. Cancer immunotherapy is one area that has yielded much recent success, although not all patients benefit equally. At the same time, recent studies have highlighted the heterogeneity of the human immune
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Staining of Frozen and Formalin‐Fixed, Paraffin‐Embedded Tissues with Metal‐Labeled Antibodies for Imaging Mass Cytometry Analysis
Qing Chang, Olga Ornatsky, David Hedley
This unit describes protocols for labeling tissue sections using combinations of metal‐tagged antibodies and an iridium‐containing DNA intercalator for analysis by imaging mass cytometry. Imaging mass cytometry (IMC) allows the labeling of up to 40 individual markers simultaneously using antibody
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Analysis of Gene‐Gene Interactions
Brian S. Cole, Molly A. Hall, Ryan J. Urbanowicz, Diane Gilbert‐Diamond, Jason H. Moore
The goal of this unit is to introduce epistasis, or gene‐gene interactions, as a significant contributor to the genetic architecture of complex traits, including disease susceptibility. This unit begins with an historical overview of the concept of epistasis and the challenges inherent in the
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Population Stratification in Genetic Association Studies
Jacklyn N. Hellwege, Jacob M. Keaton, Ayush Giri, Xiaoyi Gao, Digna R. Velez Edwards, Todd L. Edwards
Population stratification (PS) is a primary consideration in studies of genetic determinants of human traits. Failure to control for PS may lead to confounding, causing a study to fail for lack of significant results, or resources to be wasted following false‐positive signals. Here, historical and
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Analysis and Annotation of Whole‐Genome or Whole‐Exome Sequencing Derived Variants for Clinical Diagnosis
Elizabeth A. Worthey
Over the last 10 years, next‐generation sequencing (NGS) has transformed genomic research through substantial advances in technology and reduction in the cost of sequencing, and also in the systems required for analysis of these large volumes of data. This technology is now being used as a standard
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Matchmaker Exchange
Nara L. M. Sobreira, Harindra Arachchi, Orion J. Buske, Jessica X. Chong, Ben Hutton, Julia Foreman, François Schiettecatte, Tudor Groza, Julius O.B. Jacobsen, Melissa A. Haendel, Kym M. Boycott, Ada Hamosh, Heidi L. Rehm,
In well over half of the individuals with rare disease who undergo clinical or research next‐generation sequencing, the responsible gene cannot be determined. Some reasons for this relatively low yield include unappreciated phenotypic heterogeneity; locus heterogeneity; somatic and germline
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Co‐Differentiation of Human Pluripotent Stem Cells‐Derived Cardiomyocytes and Endothelial Cells from Cardiac Mesoderm Provides a Three‐Dimensional Model of Cardiac Microtissue
Elisa Giacomelli, Milena Bellin, Valeria V. Orlova, Christine L. Mummery
The formation of cardiac mesodermal subtypes is highly regulated in time and space during heart development. In vitro models based on human pluripotent stem cells (hPS cells) provide opportunities to study mechanisms underlying fate choices governing lineage specification from common cardiovascular
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Induced Pluripotent Stem Cells from Ovarian Tissue
Sophia Salas, Nicholas Ng, Behzad Gerami‐Naini, Raymond M. Anchan
Yamanaka and colleagues revolutionized stem cell biology and regenerative medicine by observing that somatic cells can be reprogrammed into pluripotent stem cells. Evidence indicates that induced pluripotent stem (iPS) cells retain epigenetic memories that bias their spontaneous differentiation into
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A Detailed Protocol for Subcellular RNA Sequencing (subRNA‐seq)
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
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Single‐Assay Profiling of Nucleosome Occupancy and Chromatin Accessibility
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
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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
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
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Production of Purified CasRNPs for Efficacious Genome Editing
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
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CASAAV: A CRISPR‐Based Platform for Rapid Dissection of Gene Function In Vivo
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
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