title: Identification of eccDNA associated with colorectal cancer metastasis by circle-seq
design: non-metastatic colorectal tumor tissuesGenomic DNA was extracted using Qiagen kits;Then, total DNA were alkaline treated to separate chromosomal DNA, lipids, and protein by rapid DNA denaturingrenaturing, followed by column chromatography on an ion exchange membrane column (Plasmid Mini AX; A&A Biotechnology);Remaining linear DNA was removed by exonuclease (Plasmid-Safe ATP-dependent DNase, Epicentre), assisted by rare-cutting endonuclease MssI (only support Homo sapiens) that digested mitochondrial circular DNA (mtDNA, 16 kb) and made additional accessible DNA ends for exonuclease;EccDNA-enriched samples was used as template for phi29 polymerase reactions (REPLI-g Midi Kit) amplifying DNA;Amplified circular DNA was cleaned (AMPure XP beads) and sheared by sonication (Bioruptor) to average fragment size of 200300 bp;Libraries for next-generation sequencing were prepared using the NEBNext Ultra DNA Library Kit for Illumina according to the manufacturers protocol (New England Biolabs), and subjected to sequencing on Illumina Novaseq 6000 using PE150.