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. 2021 May 5;11(9):e4003. doi: 10.21769/BioProtoc.4003

Figure 5. Validation of eccDNA in OVCAR8 cells by metaphase FISH.

Figure 5.

Metaphase spread of the chromosomes was carried out and the eccDNAs were identified by FISH. A representative eccDNA locus Chr2:238136071-238170279 (top row – probe 1) or Chr10:103457331-103528085 (bottom row – probe 2) was identified from OVCAR8 ATAC-seq and used for specific BAC probe design. The metaphase spreads from C4-2B cells on the left show no extrachromosomal circular DNA (negative control), while the spreads from OVCAR8 cells on the right confirm the presence of an extrachromosomal eccDNA signal (green: BAC probe, blue: DAPI). The red arrow indicates the eccDNA signals (which can be a singlet or a doublet due to replication of the eccDNA). The yellow arrows mark chromosomal DNA signals (which is usually a doublet but can be a singlet because the signal is seen from only a single chromatid).