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Transcriptomic evaluation regarding ovarian signaling at the emergence from the embryo from obligate diapause in the American mink (Neovison vison).
The gut microbiome is dominated by lysogens, bacteria that carry bacterial viruses (phages). Uncovering the function of phages in the microbiome and observing interactions between phages, bacteria, and mammalian cells in real time in specific cell types are limited by the difficulty of engineering fluorescent markers into large, lysogenic phage genomes. Here, we present a method to multiplex the engineering of life-cycle reporters into lysogenic phages and how to infect macrophages with engineered lysogens to study these interactions at the single-cell level. For complete details on the use and execution of this protocol, please refer to Bodner et al. (2020).This protocol describes immunoprecipitation of proteins associated with FLAG-tagged recombinant proteins followed by mass spectrometry-based proteomics to identify the associated interactome components. FLAG epitope was chosen, because existing high-affinity monoclonal antibodies allow for sensitive immunoprecipitation and FLAG peptides permit efficient elution of protein complexes. With many commercially available FLAG tools, this protocol is highly versatile. This procedure reduces immunoprecipitation of nonspecific binding proteins. Gene ontology analyses performed following mass spectrometry-based proteomics may elucidate novel functions of proteins of interest. selleck chemical For complete details on the use and application of this protocol, please refer to Valdez-Sinon et al. (2020).The biological phenotype is affected by the level of gene expression. Here, we provide a step-by-step protocol for precisely titrating and quantitatively observing the target gene expression level in budding yeast by manipulating its copy number in the genome. Using this method, we construct various strains with different gene copy numbers of the cell cycle inhibitor Whi5. This protocol enables stable and inherent control of gene expression at the expected level with fluorescent intensity as the quantitative readout. For complete details on the use and execution of this protocol, please refer to Qu et al. (2019).Deciphering cell cycle phases of polyphenic tissues is an important challenge in understanding the cellular mechanism of polymorphism. We use flow cytometry to analyze cell cycle phases of short wings and long wings of the brown planthopper. This provides information on the arresting cell cycle phases in different wing forms. The protocol could be applied to analysis of the cell cycle phases of other polyphenic insects and in different polyphenic tissues after modification. For complete details on the use and execution of this protocol, please refer to Lin et al. (2020).Disrupted chromatin regulatory processes contribute to the development of cancer, in particular pancreatic ductal adenocarcinoma. The assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) is typically used to study chromatin organization. Here, we present a revised ATAC-seq protocol to study chromatin accessibility in adherent patient-derived cell lines. We provide details on how to calculate the library molarity using Agilent's Bioanalyzer and an analysis pipeline for peak calling and transcription factor mapping. For complete details on the use and execution of this protocol, please refer to Brunton et al. (2020).Mammalian hematopoietic stem cells (HSCs) maintain life-long hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after bone marrow injury or in vitro culture. We have recently demonstrated the ability to maintain HSC quiescence in vitro by mimicking the bone marrow microenvironment. Here, we provide a detailed protocol for keeping functional HSCs in the quiescent state in vitro. For complete details on the use and execution of this protocol, please refer to Kobayashi et al. (2019).GLOE-Seq is a next-generation sequencing method for the genome-wide mapping of 3'-OH termini, either resulting from single- or double-strand breaks or introduced by enzymatic conversion of lesions or modified nucleotides. This protocol provides instructions for isolation of genomic DNA from budding yeast or mammalian cells, preparation of libraries for sequencing, and data analysis by the associated computational pipeline, GLOE-Pipe. It is optimized for the Illumina next-generation sequencing platform and can be adapted to intact genomic DNA of any origin. For complete details on the use and execution of this protocol, please refer to Sriramachandran et al. (2020).The peroxidase APEX2 has been used widely for proximity biotinylation and subsequent proteomics analyses. However, the poor membrane permeability of the biotin phenol substrate and the inhibitory effect of peroxide on the enzyme's activity has hampered proximity labeling in certain cell culture systems and tissues. Here, we describe an APEX2 protocol that uses alternative peroxide and biotin phenol concentrations. The protocol permits robust proximity biotinylation in confluent epithelial cell cultures and may be applicable to other cell cultures and tissues. For complete details on the use and execution of this protocol, please refer to Tan et al. (2020).The multidimensional cargo of extracellular vesicles (EV) released in urine is a reflection of the pathophysiological processes occurring within their cells and tissues of origin in the urogenital system. Here, we describe a step-by-step protocol for density-based separation of urinary EV with high specificity and repeatability. The implementation of integrative omics allows the study of the molecular complexity of highly purified urinary EV, supporting the identification of EV-specific functions and biomarkers. For complete details on the use and execution of this protocol, please refer to Dhondt et al. (2020).Analysis of mitochondrial respiration function represented by the oxygen consumption rate is necessary for assessing mitochondrial respiration function. This protocol describes steps to evaluate the respiration function of mitochondria in situ in saponin-permeabilized cardiomyocytes. In permeabilized cells, mitochondria are in a relatively integrated cellular system, and mitochondrial respiration is more physiologically relevant than isolated mitochondria. For complete details on the use and execution of this protocol, please refer to Gong et al. (2015a) and Gong et al. (2015b).
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