Targeted Sequencing of Circular RNAs for Illumina-Based Counting and Nanopore Structure Determination

Methods Mol Biol. 2024;2765:127-142. doi: 10.1007/978-1-0716-3678-7_7.ABSTRACTIn the past years, circular RNAs (circRNAs) became a major focus of many studies in animals and plants. circRNAs are generated by backsplicing from the same linear transcripts that are canonically spliced to produce, for example, mature mRNAs. They exhibit tissue-specific expression pattern and are potentially involved in many diseases, among them cardiovascular diseases. However, despite the tremendous efforts to establish circRNA catalogues, much less is known about the biological function of the vast majority of circRNAs. We have previously introduced Lexo-circSeq, a targeted RNA sequencing approach that can profile up to 110 circRNAs and their corresponding linear transcripts in one experiment from low amounts of input material on the Illumina platform. Here, we present an improved protocol for Lexo-circSeq and now extend our approach to Nanopore sequencing, which allows the structural assessment of small- and medium-sized circRNAs. Employing human-induced pluripotent stem-cell-derived cardiomyocytes originating from healthy controls or patients suffering from hypertrophic cardiomyopathy, we identify deregulated circRNAs and alternative exon usage.PMID:38381337 | DOI:10.1007/978-1-0716-3678-7_7
Source: Mol Biol Cell - Category: Molecular Biology Authors: Source Type: research