Methods in molecular biology
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The analysis of genome-wide epigenomic alterations including DNA methylation has become a subject of intensive research for many complex diseases. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies can be considered the gold standard for a comprehensive and quantitative analysis of cytosine methylation throughout the genome. Several approaches including tagmentation- and post bisulfite adaptor tagging (PBAT)-based WGBS have been devised. ⋯ Spike-in of unmethylated DNA allows for the precise estimation of bisulfite conversion rates. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human samples as well as DNA extracted from plant tissues and yields robust and reproducible results.
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Several CRISPR/Cas9 tools have been recently established for precise genome editing in a wide range of filamentous fungi. This genome editing platform offers high flexibility in target selection and the possibility of introducing genetic deletions without the introduction of transgenic sequences. This chapter describes an approach for the transformation of Penicillium chrysogenum protoplasts with preassembled ribonucleoprotein particles (RNPs) consisting of purified Cas9 protein and in vitro transcribed single guide RNA (sgRNA) for the deletion of genome sequences or their replacement with alternative sequences. This method is potentially transferable to all fungal strains where protoplasts can be obtained from.
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Increasingly, patient models of disease are being utilized to facilitate precision medicine approaches through molecular characterization or direct chemotherapeutic testing. Organoids, 3-dimensional (3D) cultures of neoplastic cells derived from primary tumor specimens, represent an ideal platform for these types of studies because benchtop protocols previously developed for 2-dimensional cell lines can be adapted for use. These protocols include directly testing the survival of these organoid cultures when exposed to clinically relevant chemotherapeutic agents, a process we have called pharmacotyping. ⋯ While our protocol has been developed for use with patient-derived pancreatic ductal adenocarcinoma organoids, with minor modifications to the dissociation and medium conditions, this protocol could be adapted for use with a wide range of organoid cultures. We further describe our standard ATP-based assay to determine cellular survival. This protocol can be scaled for use in high-throughput assays.
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DNA methylation is a covalent modification of DNA that plays important roles in processes such as the regulation of gene expression, transcription factor binding, and suppression of transposable elements. The use of whole genome bisulfite sequencing (WGBS) enables the genome-wide identification and quantification of DNA methylation patterns at single-base resolution and is the gold standard for analysis of DNA methylation. Computational analysis of WGBS data can be particularly challenging, as many computationally intensive steps are required. ⋯ Second, DNA methylation levels are estimated at each cytosine position using the aligned sequence reads of the bisulfite treated DNA. Third, regions of differential cytosine methylation between samples can be identified. Finally, these data need to be visualized and interpreted in the context of the biological question at hand.
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Exon skipping is an emerging approach to treating Duchenne muscular dystrophy (DMD), one of the most common lethal genetic disorders. Exon skipping uses synthetic antisense oligonucleotides (AONs) to splice out frame-disrupting exon(s) of DMD mRNA to restore the reading frame of the gene products and produce truncated yet functional proteins. ⋯ Although the success of multiple exon skipping in a DMD dog model has made a significant impact on the development of therapeutics for DMD, unmodified AONs such as phosphorodiamidate morpholino oligomers (PMOs) have little efficacy in cardiac muscles. Here, we describe our technique of intravenous injection of a cocktail of peptide-conjugated PMOs (PPMOs) to skip multiple exons, exons 6 and 8, in both skeletal and cardiac muscles in dystrophic dogs and the evaluation of the efficacy and toxicity.