GSM8798080: AGE2 [ChIP-Seq]; Mus; ChIP-Seq
Total DNA extraction from liver tissues was performed via column centrifugation, utilizing a technique developed by BEYOTIME. Ten micrograms of the total DNA was then subjected to a 25-unit Plasmid-Safe Dnase (Epicenter) treatment at a sustained temperature of 37°C for a 24-hour period, with the intention of eliminating linear DNA sections. Ensuing the inactivation of Plasmid-Safe DNAse, MSSI enzyme (Thermo Fisher Scientific) was introduced to eradicate mitochondrial DNA. EccDNA was subsequently recuperated via the Plasmid Recovery Kit (TIANGEN) and eluted through a 30-μl RNase-free water medium. A 50% (14 μl) fraction of the resultant eccDNA was then exposed to a 16-hour RCA reaction at 30°C.Roughly 500 ng of the amplified DNA underwent sonication, facilitated by a Covaris LE220, to generate a median fragment size of 400-bp. Further, 50 ng of the resultant fragmented DNA was employed for library construction, utilizing the MGIEasy DNA Library Preparation Kit produced by MGI-BGI, China. The quality and length distribution of each library were subsequently analyzed using an Agilent Bioanalyzer 2100 procured from Agilent. The sequencing phase was executed utilizing the MGISeq-2000 platform (supplied by Cloudseq, Shanghai, China) incorporating PE150 DNA libraries. The next phase of the study entailed the identification of eccDNA. To detect eccDNA in PE150 high-throughput sequencing data, we utilized Circle-Map (version 1.1.4) software (https://github.com/iprada/Circle-Map). Initially, a BWA-MEM alignment of sequencing reads was conducted against the mouse reference genome (mm10 genome download from UCSC). Subsequently, circular reads were extracted from two BAM files by sorting them based on their read names and coordinates. The coordinates of each eccDNA were then calculated from the aforementioned files.
GEO
GSM8798080_r1