title: GSM2402732: Nb A; Oryza sativa Japonica Group; OTHER
library_construction_protocol: Total DNA of seedlings was extracted as previously reported (Mette, M. F. et al. (1999) EMBO J 18, 241-248). Enrichment of extrachromosomal circular DNA was performed according to (Lanciano et al. submitted). 5 μg of genomic DNA for each sample were purified using a Geneclean kit (MPBio, USA) according to the manufacturer's instructions. ecDNA was isolated from the GeneClean product using the PlasmidSafe DNase (Epicentre, USA) according to the manufacturer's instructions, except that the 37°C incubation was performed for 17h. DNA samples were precipitated by adding 0.1 volume of 3M sodium acetate (pH 5.2), 2.5 volumes of ethanol and 1 μl of glycogen (Fisher, USA) and incubating overnight at -20°C. The precipitated circular DNA was amplified by random rolling circle amplification using the Illustra TempliPhi kit (GE Healthcare, USA) according to the manufacturer's instructions except that the incubation was performed for 65h at 28°C. The DNA concentration was determined using the DNA PicoGreen kit (Invitrogen, USA) using a LightCycler480 (Roche, USA). One nanogram of amplified ecDNA from each sample was used to prepare the libraries using the Nextera XT library kit (Illumina, USA) according to the manufacturer's instructions. Samples were pooled and loaded onto a MiSeq platform (Illumina, USA) and 2x250 nucleotides paired-end sequencing was performed.
identifiers.namespace: GEO
identifiers.id: GSM2402732