Indeed as Ilya says, BrdU should only be added for a single S-phase. The longer you incubate with BrdU (the more replication cycles incorporate BrdU) the less available DNA you will habe after the RASER-FISH procedure :)

Indeed difficult! Only the replicated strands are available for degradation, do ideal would be to BrdU-label in the S-phase prior to fixation. I guess for zebrafish embryos there is no uptake from outside?

BrdU incorporation during S-phase, UV-induced single-strand nicking after cell fixation and Exonuclease based digestion of the nicked DNA strand! :)

This has been an excellent collaboration together with Kai Beckwith in the @ellenberglab.bsky.social @embl.org. Thanks a lot to Kai, Jan, Natalia, and our collaborator Ralf Jungmann!!

A summary of the work can also be read at the @embl.org press release:
www.embl.org/news/science...

We thus propose that mitotic chromosome shape is dictated by Condensin-driven extrusion of long and overlapping loops up to a density set by chromatin self-repulsion.

Our model perfectly recapitulated our tracing data & the Condensin localization pattern as quantified recently in Walther et al. 2018.
rupress.org/jcb/article/...

Feature 1: Condensins pass each other. This leads to the formation of large, extensively overlapping loops with sizes up to 6-8 Mb.

Feature 2: DNA self-repulsion. This leads to self-organization and stacking of loops into the rod-shape.

Using a dynamic polymer constrained only by available quantitative Condensin chromatin-binding data, we could recapitulate the formation of native mitotic chromosome rods!

Two features were essential for that ->

We found that Condensins are also responsible for the backfolding of DNA at much larger genomic distances: up to 6-8 megabases.

Analyzing these traces, we found evidence for continuously growing loops, and our degron-based experiments showed that these loops are built by the Condensin complexes.

Kai and I used the recently established LoopTrace technology to trace the structure of single mitotic chromosomes in human cells in a very mild & structure pre-serving manner.

History will remember this day— when an American President and Vice President abandoned all we stand for.