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Jan Soroczynski

@jsoro.bsky.social

Jan Soroczynski @jsoro.bsky.social · 21/01/2026

At CTCF boundaries, walk polarity is asymmetric between flanking nucleosomes—biased toward the loop interior. Consistent with directional cohesin extrusion, resolved at nucleosome scale. CADwalks span sub-nucleosomal to chromosomal scales in a single assay. 15/

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Jan Soroczynski @jsoro.bsky.social · 21/01/2026

Analyzing fragment order in CADwalks, we find shuttling at long range – consecutive steps that venture out and back. Fragments making distal contacts are enriched for active regulatory chromatin. We also probe local fiber folding and nucleosomal footprints. 14/

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Jan Soroczynski @jsoro.bsky.social · 21/01/2026

This unlocks CADwalks: consecutive ligations between nucleosomal and sub-nucleosomal fragments, sequenced as intact concatemers. Each walk from a single nucleus. Reduced base-composition bias, no end repair. 13/

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Jan Soroczynski @jsoro.bsky.social · 21/01/2026

Because CAD-cleaved ends are natively ligatable, we can skip end repair and biotin entirely. Direct CAD-C does this and still yields high-quality maps. Native CAD ends ligate directly to each other. 12/

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Jan Soroczynski @jsoro.bsky.social · 21/01/2026

We call this ligation-gating. Capturing a loop requires both anchors in proximity AND both ligatable at once. CAD's ligation-competent ends raise this joint probability –consistent with both the loop sensitivity and the short-range enrichment. See Supplementary Note. 11/

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Jan Soroczynski @jsoro.bsky.social · 21/01/2026

But here's the puzzle: 1D ligation-junction coverage at loop anchors looks nearly identical between CAD-C and Micro-C. So why more loops? It's not about cutting more at accessible sites. It's about both anchors being simultaneously ligatable in the same cross-linked complex. 10/

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Jan Soroczynski @jsoro.bsky.social · 21/01/2026

CAD-C recapitulates compartments, TADs, & loops – w/ same or improved contrast. CAD-C-detected loops skew toward active chromatin; we see more TSS-anchored loops than Micro-C or Hi-C. CAD-C detects promoter loops to CRISPRi-validated enhancers that Micro-C misses at matched depth. 9/

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Jan Soroczynski @jsoro.bsky.social · 21/01/2026

We tested CAD-C in three human cell lines at saturating CAD concentrations. All worked on first try. Ligation efficiency was high, producing long concatemers. 8/

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Jan Soroczynski @jsoro.bsky.social · 21/01/2026

We optimized the CAD:ICAD construct for purification from E. coli and orthogonal activation by TEV protease. Result: consistent genome coverage across a 125-fold CAD concentration range. 7/

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Jan Soroczynski @jsoro.bsky.social · 21/01/2026

Enter CAD (DFF40), a metazoan apoptotic nuclease. It has what we needed: reduced base-composition bias vs MNase, strict endonuclease (no chew-back), and ligation-ready ends (blunt 5'-P/3'-OH). CAD is naturally held inactive by its chaperone-inhibitor ICAD. 6/

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