Circle-Seq for extrachromosomal circular DNA in PLC-PRF-5 cells after treatment with 25OHD

It has been verified that extrachromosomal circular DNA (ecDNA) has important clinical significance, and the formation process of ecDNA, its immunogenicity, and its other functions have also been described. However, the balancing mechanism by which cells respond to ecDNA, i.e., how ecDNA degrades within cells, is unclear. Here, by using Circle-Seq method conducted on PLC-PRF-5 HCC Cells, ecDNA was purified and the samples were used for DNA Library Preparation and Next Generation Paired-End Sequencing using Illumina MiSeq Technology, We found that some ecDNAs disappeared after VD treatment compared with the DMSO group, and these disappearing ecDNA were related to EMT, proliferation, cell cycle, focal adhesion and other malignant behaviors. Moreover, Circle-seq peaks narrowed after VD treatment, and the coverage over the whole gene was significantly reduced, suggesting that ecDNA after VD treatment carried smaller DNA fragments, which may cause them not to function as a whole gene. Importantly, we demonstrate that VD can cause a continuous and steady decrease in ecDNA in HCC cells. We purified ecDNA from PLC-PRF-5 cells treated with DMSO or 25OHD (100nM and 500nM) for 48 h owing to the remarkably higher level of ecDNA content among HCC cell lines. The obtained genomic DNA was treated with Plasmid-Safe ATP-dependent DNase to eliminate linear chromosomal DNA and then DNAs were eluted and digested with PacI to eliminate mitochondrial DNA. we employed rolling-circle amplification of the purified eccDNA by MDA using φ29 DNA polymerase and random hexamer primers. We than sequenced the purified eccDNA using Illumina paired-end sequencing.