SNU16-CircleSeq
Circular DNA was harvested from the human cancer cell lines described above using Monarch HMW DNA Extraction Kit (NEB, #T3050). The linear chromosomal DNA was digested by plasmid-safe ATP-dependent DNase (Lucigen, E3101K) with 25mM ATP and provided reaction buffer. The reaction was incubated at 37 C overnight, and the DNase was heat-inactivated by incubating at 70C for 30min. Circular DNA was next purified using an eccDNA purification kit (CAT#:220501-50), then amplified using the REPLIg Midi Kit (QIAGEN, 150043) according to the manufacturers instructions. Amplified circular DNA was measured with a Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and sheared to an average fragment size of 150200bp. Libraries were prepared using the NEBNext Ultra DNA Library Kit for Illumina according to the manufacturers protocol (New England Biolabs) and sequenced with 2150-bp paired-end reads on the Illumina NovaSeq 6000 system according to the manufacturers instructions, with an average data volume of 30G reads.