GSM5058061: mESC purified eccDNA RCA nanopore; Mus musculus; OTHER
Nanopore sequencing library for eccDNA was prepared by following Ligation Sequencing Kit (Oxford Nanopore) according to manufactory's instruction after rolling cycle amplification and debranching. RCA were performed with phi29 DNA polymerase (NEB) with some modification to ensure efficient amplification from 100 pg template per reaction. Briefly, in a 20 μl reaction mixture: 2 μl 10X phi29 DNA polymerase buffer (NEB), 2 μl 25 mM dNTPs, 1 μl Exo Resistant Random Primer (Thermo Fisher), and eccDNA >=100 pg, were added with ultra-pure H2O to 17.6 μl, mixed and incubated at 95 ˚C for 5 minutes before being ramped to 30 ˚C at 1% Ramp Rate. Then, 1 μl phi29 DNA polymerase, 0.6 μl Pyrophosphatase Inorganic (yeast, NEB), 0.4 μl 0.1M DTT (NEB), 0.4 μl 20 mg/ml BSA (NEB) were added. The reaction mixture was incubated at 30 ˚C for 10-16 hours. Since high branch structure of RCA products could block nanopore to abolish sequencing, RCA products of eccDNAs were further debranched with T7 Endonuclease I (NEB) before being used for sequencing library construction with Ligation Sequencing Kit (Oxford Nanopore, SQK-LSK109). The library was sequenced in Flow cell (R9.4.1, FL-MIN106D) on MinION according to manufactory's instruction. 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