Illustra templiphi kit

The sequencing of the eccDNA from Drosophila embryos was done as described in (35 (link)). Briefly, genomic DNA was extracted from 0–2 h embryos using the DNeasy Blood & Tissue Kit (Qiagen) and further purified with the PCR purification kit (Qiagen). Linear genomic DNA was digested with Plasmid-Safe™ ATP-Dependent DNase (Lucigen) and the remaining eccDNA molecules were then amplified by rolling circle amplification (RCA) using Illustra TempliPhi kit (GE Healthcare) and random primers. Sequencing libraries were prepared with the Nextera XT library kit (Illumina) and 250 nucleotides paired-end sequencing was performed using the MiSeq platform (Illumina) by Plateau de Génotypage CIRAD (France). For PCR confirmation 10 ng of RCA amplified DNA was used (for primer sequences see Supplementary Table S2).

The 25 µL total DNA isolated from plant or nematode tissues was treated with PlasmidSafe DNase (Epicentre) following the manufacturer's instructions, except the reaction was incubated at 37 °C for 17 h. Digested DNA samples were cleaned and precipitated following ethanol as described previously, except the DNA pellet was resuspended in 5 µL of TempliPhi sample buffer instead of water. eccDNA as well as other circular DNAs was amplified from the resuspended DNA solution through RCA by using the Illustra TempliPhi Kit (GE Healthcare). The reaction was performed according to the manufacturer's instructions, and the incubation for RCA was performed at 28 °C for 72 h to maximize the amplification. The Phi29 enzyme used for RCA reaction was deactivated by incubating the sample at 70 °C for 30 min. For both A. thaliana and H. schachtii samples (listed in supplementary table S3, Supplementary Material online), 400 ng of input DNA was sent to Novogene, Cambridge, United Kingdom, for 150 bp paired-end TruSeq Library preparation and sequencing. DNA was performed on a NovaSeq 6000 (Illumina).