(7/13) Armed with this information, I then tested whether hypotonic conditions and HDAC inhibition untether ecDNA from chromosomes in cells cultured on glass coverslips with better morphological preservation. Indeed they did.

(6/13) When I tested this by replacing the hypotonic solution (75 mM KCl) incubation step with HDAC inhibitor (TSA) treatment, which is known to decompact mitotic chromatin, very nice prometaphase spreads were produced with separated chromosomes (DAPI) and ecDNA (MYC).

(5/13) In spreads of ecDNA-containing cells, the ecDNA are also separated and untethered from chromosomes. I hypothesized that the hypotonic solution incubation step of the preparation disrupts the electrostatic or hydrophobic forces that compact chromatin during mitosis.

(4/13) I became interested in prometaphase spread preparations of cells, in which all the chromosomes are nicely distinct and separated from each other. However, how exactly prometaphase spread preparations achieve this is not clearly described. (Image from Wikipedia)

(3/13) I started with a simple hypothesis: ecDNA-chromosome tethering is not an ecDNA-specific phenomenon. Rather, maybe all chromatin tether to each other during mitosis, including chromosomes with each other, as chromatin should be quite sticky during mitotic compaction.