title: eccDNA of Arabidopsis
design: Genomic DNA was extracted from the Arabidopsis tissue samples using the Qiagen Plant DNeasy mini kit (Qiagen), and DNA concentrations were recorded using Nanodrop (Thermo Scientific). DNA samples with concentrations more than 10 ng/l and OD260/280 values in between 1.8-2.2 were combined and used for further experiment. For each sample, 3.0ug of genomic DNA was used for the removal of large genomic linear fragments using a Geneclean kit (MPBio), and the protocol was according to the manufacturers instructions. The GeneClean products were digested with the ATP-dependent PlasmidSafe DNase I kit (Epicentre) to remove the remaining linear DNA fragments, by adding 7.5l 10x reaction buffer, 3l (25mM) ATP, 15units (1.5l) DNase I and 35l H2O to 28l eluted DNA to reach a final volume of 75l. The incubations were performed at 37oC for at least 3 days, and every 24 hours 0.5 l 10x reaction buffer, 3l ATP(25mM) and 15 units of DNase I were added to maintain continuous enzymatic digestion. Complete removal of linear DNA was confirmed with polymerase chain reaction (PCR), using a chromosomal marker- the ACTIN2 specific primers 5-GCACCCTGTTCTTCTTACCG-3 and 5-AACCCTCGTAGATTGGCACA-3. For a 20l of PCR reaction, 1l DNase I treated sample, 5 pic moles of forward and reverse primers each, and 10ul of PCR reaction mix(TIANGEN BIOTECH, Beijing) were added. PCR reaction condition was as the following: 3 minutes at 95oC; followed by 35 cycles of 30sec at 95oc, 30sec at 58oC, 30sec at 72oC. PCR reactions were analyzed by gel electrophoresis on 2% agarose with 0.5ug/ml ethidium bromide. Complete removal of linear chromosomal DNA was indicated as the absence of 283bp- ACTIN2 amplification band. Upon removal of linear chromosomal DNA, the DNA samples were precipitated by adding 0.1 volume of 3M NaAc (pH5.2), 2.5 volumes of ethanol and 1l of glycogen (Fisher) and incubating overnight at -80oC, and eccDNA were resuspended in 20ul TE buffer (pH 8.0). The purified eccDNA samples were amplified by random RCA using the REPLI-g Mini Kits (Qiagen), according to the protocol provided by the polymerase manufacturer. The RCA incubation was performed at 30oC for 72 hours. At the end of the reaction 1l of each amplified DNA sample was loaded on an agarose gel (1%) to confirm successful RCA amplification. The amplified eccDNA samples were sent to GENERGY BIO Company (Shanghai) for library preparation and deep sequencing. In brief, from each sample 0.5ug of the amplified eccDNA was used to prepare the libraries using the TruSeq DNA LT Sample Prep Kit v2 (Illumina), and the protocol was according to the manufacturers user guide. Each mobilome-seq library was amplified by 10 cycles of PCR using index primers. DNA qualities and concentrations were then determined by using Qubit picogreen (PicoGreen) and via 1% agarose gel electrophoresis(120V, 15min). Samples were pooled and loaded onto a flow cell and 2x150 necleotides paired-end sequencing was performed using the HiSeq3000 platform (Illumina) using Hiseq3000SBS&Cluster kit(Illumina). Up to twelve mobilome-seq libraries were pooled into one run and an average of 1 million reads per library were obtained. Illumina reads were collected for the analysis as FASTQ files.