GSM5058062: mESC purified eccDNA illumina; Mus musculus; OTHER
Illumina sequencing library for eccDNA was prepared by Tn5-transposon-based tagmentation with Nextera® XT DNA Sample Preparation Kit according to manufactory's instruction. Briefly, after validating the purity of eccDNAs with AFM imaging, 0.5 ng pure eccDNAs were directly tagmented with Tn5 transposase, followed by 12-14 cycles PCR amplification with Illumina sequencing adaptors. Barcoded libraries were pooled and sequenced with Illumina HiSeq 2500 in 150 PE mode. To purify eccDNAs, cells were first dehydrate in >90% methanol before crude extrachromosomal DNA were extracted in an alkaline lysis buffer at pH11.8. After neutralization and precipitation, crude extrachromosomal DNA were bound in silica column (Qiagen) in binding buffer. Bound DNAs were eluted and digested with PacI (NEB) and Plasmid Safe ATP-dependent DNase (Lucigen) for 4-16 hours. Undigested eccDNA is then extracted with Phenol/chloroform/isoamyl alcohol (PCI) solution (25:24:1) in a Phase Lock Gel tube (QuantaBio) to minimize DNA loss. After precipitation with carrier glycogen (Roche) and 1/10 V 3M NaOAc (pH 5.5), the precipitated crude eccDNAs were resuspended in Solution P and further purified by magnetic silica beads that selectively bind to eccDNAs. The eccDNAs were then eluted with 0.1X Elution buffer (1mM Tris-HCl pH8.0) and the concentration is measured by Qubit dsDNA HS Assay kit (Thermo Fisher). purified eccDNA seq