title: E1
design: For samples of tissue and cell, samples of tissue or cell pellets were suspended in L1 solution (Plasmid Mini AX; A&A Biotechnology) and supplemented with Proteinase K (ThermoFisher) before incubation overnight at 50 C with agitation. After Lysis, samples were alkaline treated,followed by precipitation of proteins and separation of chromosomal DNA from circular DNA through an ion exchange membrane column (Plasmid Mini AX; A&A Biotechnology). Column-purified DNA was treated with FastDigest MssI (Thermo Scientifific) to remove mitochondrial circular DNA and incubated at 37 C for 16 h. Remaining linear DNA was removed by exonuclease (Plasmid-Safe ATP-dependent DNase, Epicentre) at 37 C in a heating block and enzyme reaction was carried out continuously for 1 week, adding additional ATP and DNase every 24 h (30 units per day) according to the manufacturers protocol (Plasmid-Safe ATP-dependent DNase, Epicentre). eccDNA-enriched samples was used as template for phi29 polymerase amplification reactions (REPLI-g Midi Kit) amplifying eccDNA at 30 C for 2 days (4648 h).Phi29-amplifified DNA was sheared by sonication (Bioruptor), and the fragmented DNA was subjected to library preparation with NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs). Sequencing was carried out on Illumina NovaSeq 6000 with 150bp paired end mode according to the manufacturers instructions.