GSM7731084: NF3; Homo sapiens; OTHER
samples of tissue 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 manufacturer's protocol (Plasmid-Safe ATP-dependent DNase, Epicentre). eccDNA-enriched samples were used as template for phi29 polymerase amplification reactions (REPLI-g Midi Kit) amplifying eccDNA at 30 °C for 2 days (46–48 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).