All reads are pair-end sequenced, measuring precise footprint of chromatin components. We identified abundant Overlapping Di-Nucleosomes (OLDN) at a subset of divergent promoters. This is supported by a single-molecule analysis showing that adjacent nucleosomes and OLDN are mutually exclusive.
10/11

Something surprising!

Not all fuzziness is due to transcription.
Rather, the genomic distance between two consecutive NFRs (Nuc Free Region) predict fuzziness. If the size is a multiple of the optimal Nucleosome Repeat Length (NRL) => Positioned Array. If not => Delocalized Array.

9/11

On very highly expressed genes, long arrays disappear, and nucleosomes typically exist in pairs. Shutting down transcription resolves the fuzziness on some genes, re-establishing nucleosomes arrays.

8/11

On short range interaction, PCP will tag nucs from the same fiber with the same UMI. This reveals that “fuzzy” nucs in classical MNase-seq profiles correspond to well-spaced nucleosome arrays slightly offset from cell-to-cell.

7/11

Interaction matrix sometimes show signal between three consecutive cohesin peaks, suggesting multiway interaction. But Micro-C can only detect pairwise interactions. With PCP we show that cohesin tends to form hubs of multiway interactions.

6/11

We can bin the results in classical interaction matrices. On S. cerevisiae, PCP perform as well as Micro-C (i.e. detecting Cohesin loops in G2). At high-resolution PCP performs better and provide a more detailed picture of nucleosome interactions.

5/11

We used PCP to map chromatin structure:

Chromatin is X-linked and digested to mononucs, a mixture of Seeds and Receptors is ligated to DNA ends. Each seed will produce unique tagRNAs and tag nucs in proximity.

After sequencing, reads are grouped based on the UMI, informing on spatial proximity.

The new method is based on a simple reaction:

• A Seed is transcribed to produce a tagRNA carrying: a unique identifier (UMI) and an annealing sequence.
• The tagRNAs diffuse and anneal to Receptors sharing the annealing sequence
• The annealed tagRNA is copied by RT to “tag” the Receptor

3/11

Hi everyone!

The latest method from the Whitehouse lab: PCP


PCP uses a novel proximity barcoding strategy to simultaneously map 3D genome organization at various resolutions at the single molecule level:

Out soon, check the updated BioRxiv – little thread:

1/11
www.biorxiv.org/content/10.1...

Cool lineup next Monday at NYU on 3D chromatin architecture and cancer.
Can follow on zoom here: nyulangone.zoom.us/webinar/regi...