Fig. 7
Identification and mapping of eccDNAs derived from the single-cell clone 1C3-1 and 6C7 by Circle-Seq. A EccDNA size distribution and mapping of eccDNAs to genic and intergenic regions in 1C3-1 (top) and 6C7 (middle) as well as in the control NHH-3T3 cell line (bottom). Total number of eccDNAs are indicated in parentheses next to the name of the cell line. Pie charts depict the distribution and percentage of eccDNAs mapped to different genic and intergenic regions in the three cell lines. Percentages of eccDNAs are also shown for genic regions that include promoters, exons, 5′-UTR and 3′-UTR. B Distribution of eccDNAs neighboring TSG target sites in various distances indicated by log10 values from distal and proximal junction point to TSG target sites in NIH-3T3, 1C3-1, and 6C7 cell lines as indicated. EccDNAs that are close to TSG target sites are also denoted by arrows and names. X and Y in the schematic (top) represent the distance from the distal and proximal junction point of eccDNAs on the chromosome to the breakpoint of TSG target site by CRISPR/Cas9, respectively. C Schematic for circularization of Apob-2119 eccDNA from chromosome 12 (Chr 12). Likely due to cleavage of the Apob target site by CRISPR/Cas9, the left end was processed and circularized via neighboring microhomology (MH) in the same chromosome to form Apob-2119 eccDNA. The four primers F1, R1, F2, and R2 indicated were designed to form one pair of inward primers F2/R2 and two pairs of outward primers R1/F1 and R2/F1 for validation of Apob-2119 eccDNA. The length of the eccDNA and the position of Apob target site by CRISPR/Cas9 are shown with the distal point of the eccDNA set at 0 bp. D Sequences of Apob-2119 eccDNA and its junctions as shown are determined by PCR with indicated primer pairs followed by Sanger sequencing. Circularization junction is also indicated