GSM6206867: Cerebrospinal fluid, Medulloblastoma, Patient2; Homo sapiens; OTHER
For samples of cerebrospinal fluid, cerebrospinal fluid DNA extractions were performed using QIAamp Circulating Nucleic Acid Kit (Qiagen). DNA was digested with exonuclease V (New England Biolabs) at 37℃ for 5 min to remove linear DNA.. The circular structure of eccDNA is opened by transposable enzymesand the ends of the DNA fragments are attached to the joints. To fill these gaps and ends, the Klenow enzyme was used for gap/end repair. Then the products were amplified and purified by PCR. For samples of tissue, samples 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 was used as template for phi29 polymerase amplification reactions (REPLI-g Midi Kit) amplifying eccDNA at 30 °C for 2 days (46–48 h). Illumina sequencing libraries were generated with NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs) by following the manufacturer's manual. The library quality was determined by using Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., USA).Sequencing was carried out on Illumina NovaSeq 6000 with 150bp paired end mode according to the manufacturer's instructions.