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Protocol
Efficient Generation of CRISPR/Cas9-Mediated Knockout Human Primary Keratinocytes by Electroporation
Due to their full differentiation capacity in vitro, the culture of human primary keratinocytes (HPKs) represents a physiological model for answering basic biological and dermatological research questions, inc...
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Protocol
Phase Separation of Phytochrome B in HEK293T Cells
In this method, we employed HEK293T cells to express the plant photoreceptor phytochrome B (phyB). Through the application of various treatments such as phycocyanobilin (PCB) supplementation, red light exposur...
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Protocol
Monitoring Real-Time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism
RNA molecules play crucial roles in gene expression regulation and cellular signaling, and these functions are governed by the formation of RNA secondary and tertiary structures. These structures are highly dy...
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Protocol
On-Chip Magnetic Extraction of Circulating Cell-Free DNA from Biological Samples
In recent years, the analysis of circulating cell-free DNA (cfDNA) containing tumor-derived DNA has emerged as a noninvasive means for cancer monitoring and personalized medicine. However, the isolation of cfD...
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Protocol
Decellularized Extracellular Matrix for Modeling Cardiac Extracellular Microenvironment
The extracellular matrix (ECM) forms most of the tissue microenvironment and is in a constant and dynamic equilibrium with cells. The decellularization process employs physical or chemical methods, or a combin...
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Protocol
High-Throughput Spectroscopic Analysis of mRNA Cap** Level
Eukaryotic mRNAs are characterized by terminal 5′ cap structures and 3′ polyadenylation sites, which are essential for posttranscriptional processing, translation initiation, and stability. Here, we describe a...
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Protocol
Characterization of Nuclear HIV-Induced Membraneless Organelles Through Fluorescence Microscopy
The postnuclear entry steps of HIV-1 involve reverse transcription, uncoating, and integration into the host genome. The differential regulation of these steps has a significant impact on HIV overall replicati...
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Protocol
The Study of Anticipatory Brain Activity in Cognitive Tasks by Means of Event-Related Potential, Frequency, and Time-Frequency Methods
This chapter presents a series of studies on anticipatory brain activity in cognitive tasks acquired using electroencephalographic methods including event-related potential, frequency, and time-frequency analy...
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Protocol
Generation of Connexin-Expressing Stable Cell Pools
Stable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressi...
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Protocol
Characterizing Post-transcriptional Modifications of circRNAs to Investigate Biogenesis and Translation
Recent studies have shown that circular RNAs (circRNAs) are decorated with N6-methyladenosine (m6A), a co-transcriptional modification known to participate in the regulation of many processes governing linear RNA...
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Protocol
Genetically Encoded Noncanonical Amino Acids in Proteins to Investigate Lysine Benzoylation
Posttranslational modifications (PTMs) of lysine residues are major regulators of gene expression, protein–protein interactions, and protein localization and degradation. Histone lysine benzoylation is a recen...
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Protocol
Single-Molecule Imaging of Enzymatic Reactions on DNA Origami
The interaction between enzymes is very important for understanding of enzyme functions and shed light on enzymatic reaction mechanisms. With the development of DNA nanotechnology, DNA origami has become a pow...
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Protocol
Analysis of Polycomb Epigenetic Marks in HeLa Spheroids
Biochemical composition of the nucleus affects both its physical properties and its morphology. In recent years, several studies demonstrated the formation of f-actin in the nuclei. These filaments intermingle...
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Protocol
Live-Cell Visualization of Early Stages of Root Colonization by the Vascular Wilt Pathogen Fusarium oxysporum
Fungal phytopathogens induce a variety of pathogenicity symptoms on their hosts. The soilborne vascular wilt pathogen Fusarium oxysporum infects roots of more than 150 different crop species. Initial colonization...
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Protocol
Discrimination of Multidrug Resistance in Cancer Cells Achieved Using Single-Cell Analysis
The biophysical signatures of single cells, such as multidrug resistance (MDR), may easily change during their various disease states. Therefore, there is an ever-growing need for advanced methods to study and...
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Protocol
Visualize the Distribution and Dynamics of Mitochondrial DNA (mtDNA) Nucleoids with Multiple Labeling Strategies
Mitochondrial DNA (mtDNA) encodes a variety of rRNAs, tRNAs, and respiratory chain complex proteins. The integrity of mtDNA supports the mitochondrial functions and plays an essential role in numerous physiolo...
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Protocol
Isolation of Chromatin Proteins by Genome Capture
Control of gene expression and the faithful transmission of genetic and epigenetic information rely on chromatin-bound proteins. These include the polycomb group of proteins, which can display a remarkable var...
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Protocol
Reverse Vaccinology for Influenza A Virus: From Genome Sequencing to Vaccine Design
Reverse vaccinology (RV) consists in the identification of potentially protective antigens expressed by any organism starting from genomic information and derived from in silico analysis, with the aim of promo...
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Protocol
PicoShells: Hollow Hydrogel Microparticles for High-Throughput Screening of Clonal Libraries
Microfluidics enables the creation of monodisperse, micron-scale aqueous droplets, or other compartments. These droplets serve as picolitre-volume reaction chambers which can be utilized for various chemical a...
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Protocol
In Vivo Study of Monoamine Oxidases Using Multisite Intracerebral Microdialysis
The activity of monoamine oxidases (MAOs) in the brain is often associated with neurodegenerative diseases. The study of MAOs in vivo or ex vivo is generally performed using MAO inhibitors and rarely using sub...
