Chromosome-derived extrachromosomal circular DNA elements (eccDNAs) are detected in all eukaryotes examined so far. Here I describe the Circle-Seq protocol, applicable for physical enrichment of eccDNAs of a broad size range, combined with sequence confirmation of circular structures. Briefly, by concise alkaline treatment and gentle gravity flow-through an ion-exchange column, eccDNAs are enriched in the eluate fraction. EccDNAs are enzymatically isolated by extensive Plasmid-Safe DNase digestion of linear chromosomes and further enriched by φ29 rolling circle amplification. By means of high throughput sequencing of amplified eccDNA and custom eccDNA mapping software, around ten-thousand unique eccDNA types could be detected at nucleotide resolution in a million human muscle nuclei by this method.
CITATION STYLE
Møller, H. D. (2020). Circle-Seq: Isolation and Sequencing of Chromosome-Derived Circular DNA Elements in Cells. In Methods in Molecular Biology (Vol. 2119, pp. 165–181). Humana Press Inc. https://doi.org/10.1007/978-1-0716-0323-9_15
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