Plants grown on ZA containing MS medium
For eccDNA isolation we used the techniques described by Lanciano et al. [51] and Wang et al. [60] with modifications. Briefly, to remove linear DNA, 1 g of total DNA was treated with 1 l (10 U/l) of PlasmidSafe DNase supplemented with 2 l of ATP (25 mM) and 5 l of 10 PlasmidSafe buffer in a volume of 50 l. The reaction was incubated for 72 hours with additional reagents (0.1 l enzyme, 0.2 l ATP, 0.3 l buffer) was added every 24 h, followed by incubation at 72C for 30 min. Precipitation of eccDNA was carried out by overnight incubation at -20C in the presence of 0.1 volume of 3 M sodium acetate (pH 5.2) and 2.5  volume of absolute ethanol, followed by centrifugation at 12,000  g for 30 min. The eccDNA pellet was washed with ice-cold 70% ethanol and dissolved in 10 l of deionized nuclease-free water. For eccDNA amplification using random RCA, 2 L phi29 polymerase (Thermo Scientific, EP0091), 2 L 10 phi29 reaction buffer, 5 L 10 mM dNTP, and 1 L 500 M exo-resistant oligo (NpNpNpNpNpSNpSN, where p is phosphodiester and pS is the phosphorothioate group) with the addition of nuclease-free water to a final volume of 20 L. The reaction was preheated to 95C for 5 min, ramped to 30C at a 1% ramp rate on a thermocycler, and incubated for 36 h at 30C. The enzyme was inactivated by heating the mixture at 65*C for 10 min. For debranching, 500 ng of RCA amplicons were treated with T7 endonuclease 5 L of 10 reaction buffer and 1 L of T7 endonuclease I (New England Biolabs, M0302S) in a 50 L reaction volume. After incubation at 37C for 15 min, the reaction was stopped immediately and purified by adding an equal volume of chloroform. The Debranched RCA product was precipitated by adding 1/10V 3M sodium acetate (pH 5.2) and absolute ethanol (2.5V), followed by incubation at 80C for 30 min and centrifugation at 12,000 g for 30 min. The pellet obtained was dissolved in nuclease-free water and used for nanopore sequencing.