Ecc_DNA identificaiton based on circle-seq for hydroquinone indcued malignant transformaned TK6 cells

The hydroquinone-induced malignantly transformed TK6 cells (HQ-TK6) were derived from TK6 cells treated with HQ for 20 weeks and termed HQ-TK6 cells. TK6 cells treated with PBS were the control cells and termed PBS-TK6 cells. The procedure for establishing HQ-TK6 cells was described in our previous report [1]. Cells were collected and lysed to obtain purified high-molecular-weight (HMW) DNA using a MagAttract HMW DNA Kit (Qiagen, 67563), and DNA quality was determined with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). To eliminate linear DNA and enrich the eccDNA, DNA was subjected to digestion with Plasmid-Safe ATP-Dependent DNase (20 units). Then, DNase and ATP were added, and the mixture was digested for at least 5 days until the digestion was complete.The efficacy and quality of eccDNA enrichment were determined by qPCR. The eccDNA-enriched DNA samples were amplified using phi29 polymerase with random hexamer oligos according to the instructions. The phi29-amplified DNA products were subsequently sheared with a focused ultrasonicator (Bioruptor, Diagenode) to generate fragments of 300-500 base pairs, followed by purification using a MinElute PCR Purification Kit (Qiagen, 28004). The fragmented DNA samples were subjected to sequencing library construction (VAHTS Universal DNA Library Prep Kit for Illumina V3, VAHTS, ND607). The library was purified by purification beads, and the size distribution of the fragments was analyzed on an Agilent 2100 Bioanalyzer (Agilent). Next-generation sequencing was performed on an Illumina NovaSeq 6000 platform using the constructed libraries. All of the libraries were sequenced as 2*150-bp paired-end reads. FastQC software was used to evaluate the quality of the original data. Low-quality bases and adapter sequences were trimmed off by Cutadapt[2], and Burrows-Wheeler Aligner software[3] was subsequently used to compare and map the original data to the reference genome (hg19). SAMtools was applied to process the SAM file to fit the format required by Circle-MAP [4]. The identified eccDNAs were subjected to annotation.References1. Mol Carcinog 2017, 56,651-663.2 J Comput Biol 2017, 24,1138-1143.3. Bioinformatics 2009, 25,1754-1760.4. Bmc Bioinformatics 2019, 20,663.