Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing <em>In Vitro</em> and in Cells
نویسندگان
چکیده
The role of RNA structure in virtually any biological process has become increasingly evident, especially the past decade. However, classical approaches to solving structure, such as crystallography or cryo-EM, have failed keep up with rapidly evolving field and need for high-throughput solutions. Mutational profiling sequencing using dimethyl sulfate (DMS) MaPseq is a sequencing-based approach infer from base's reactivity DMS. DMS methylates N1 nitrogen adenosines N3 cytosines at their Watson-Crick face when base unpaired. Reverse-transcribing modified thermostable group II intron reverse transcriptase (TGIRT-III) leads methylated bases being incorporated mutations into cDNA. When resulting cDNA mapping it back reference transcript, relative mutation rates each are indicative "status" paired Even though reactivities high signal-to-noise ratio both vitro cells, this method sensitive bias handling procedures. To reduce bias, paper provides protocol treatment cells transcribed RNA.
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ژورنال
عنوان ژورنال: Journal of Visualized Experiments
سال: 2022
ISSN: ['1940-087X']
DOI: https://doi.org/10.3791/64820-v