Methods in molecular biology
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based technology enables efficient and precise perturbations of target genomic sites. Combining the endonuclease Cas9 and a pooled guide RNA library allows for systematic screenings of genes associated with a growth disadvantage or lethal phenotype under various conditions in organisms and tissues. Here, we describe a complete protocol for scalable CRISPR/Cas9-based dropout screening for essential genes from focused genomic regions to whole genomes.
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DNA methylation is a process by which methyl groups are added to cytosine or adenine. DNA methylation can change the activity of the DNA molecule without changing the sequence. Methylation of 5-methylcytosine (5mC) is widespread in both eukaryotes and prokaryotes, and it is a very important epigenetic modification event, which can regulate gene activity and influence a number of key processes such as genomic imprinting, cell differentiation, transcriptional regulation, and chromatin remodeling. ⋯ A number of different quantitative approaches have also been established to map the DNA epigenomes with single-base resolution, as represented by the bisulfite-based methods, such as classical bisulfite sequencing, pyrosequencing etc. These methods have been used to generate base-resolution maps of 5mC and its oxidation derivatives in genomic samples. The focus of this chapter is to provide the methodologies that have been developed to detect the cytosine derivatives in the genomic DNA.
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Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) system has emerged as an extremely useful tool for biological research and as a potential technology for gene therapy approaches. CRISPR/Cas mediated genome editing can be used to easily and efficiently modify endogenous genes in a large variety of cells and organisms. ⋯ This chapter provides an introduction to the basis of the technology and a detail protocol for the most classic application: gene inactivation by CRISPR/Cas9 nuclease system from Streptococcus pyogenes. This workflow can be easily adapted for other CRISPR systems and applications.
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DNA methylation is a conserved epigenetic modification of animal genomes, but genome methylation patterns appear surprisingly diverse in insects. Whole-genome bisulfite sequencing (WGBS) represents a sensitive and robust method for the characterization of genome-wide methylation patterns at single-base resolution. Here, we describe a step-by-step protocol for the generation and analysis of WGBS datasets using standard Illumina sequencing platforms. In comparison to whole-genome sequencing, WGBS has additional caveats that require particular attention and are highlighted in this chapter.
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Despite advances in intensive care unit interventions, including the use of specific antibiotics and anti-inflammation treatment, sepsis with concomitant multiple organ failure is the most common cause of death in many acute care units. In order to understand the mechanisms of clinical sepsis and develop effective therapeutic modalities, there is a need to use effective experimental models that faithfully replicate what occurs in patients with sepsis. Several models are commonly used to study sepsis, including intravenous endotoxin challenge, injection of live organisms into the peritoneal cavity, establishing abscesses in the extremities, and the induction of experimental polymicrobial peritonitis via cecal ligation and puncture (CLP). Here, we describe the surgical procedure of CLP in mice, which has been demonstrated to closely replicate the nature and course of clinical sepsis in human subjects.