GEO series

CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells [Cut & Tag]

GSE318107 Homo sapiens Genome binding/occupancy profiling by high throughput sequencing 10 samples Submitted 2026/08/07 Platform GPL30882

Summary

Chromosome conformation capture (3C)-derived methods have become an indispensable tool in the study of gene regulation. The three-dimensional contacts can probe depend strongly on the properties of the enzyme used to fragment chromatin prior to proximity-driven ligation. Micrococcal nuclease (MNase), used in Micro-C, increases resolution at the expense of low ligation efficiency and the need for extensive enzyme titration. To overcome these limitations, we engineered a highly active, TEV protease-activatable caspase-activated DNase (CAD) to enable an efficient, low-sequence-bias, and high-resolution proximity ligation assay we call CAD-C. CAD-C was successful on the first attempt for each human cell line tested and the resulting datasets capture loops, TADs, compartments, and stripes similarly to Micro-C. However, compared to Micro-C and Hi-C, CAD-C shows enhanced sensitivity for promoter-enhancer loops. Leveraging the ligation-competent DNA ends produced by CAD cleavage, we show that CAD-C is compatible with a highly streamlined, repair-free protocol and produces multi-step CADwalks, consecutive ligations between nucleosomal or sub-nucleosomal fragments. With these walks, we probe local chromatin fiber folding contacts, nucleosomal and sub-nucleosomal footprints, and long-range nuclear organization regimes in human cell lines. CAD-C is an efficient, robust chromatin structure assay that can span sub-nucleosomal to chromosomal length scales in a single experiment.

Published in

CAD-C: An engineered nuclease enables repair-free in situ proximity ligation and nucleosome-resolution chromosome walks in human cells

Soroczynski J, Westcott LA, Zuo W et al. · bioRxiv : the preprint server for biology 2026 · PMID 42146372 · doi:10.64898/2025.12.22.695891

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Direct links to NCBI, no account and no request form: the whole study as GSE318107_RAW.tar, processed values as the series matrix, the supplementary file directory, and per-sample supplementary files for any of the 10 samples. Raw sequencing reads are also available from ENA.

Whole study · RAW.tar ↓ Series matrix ↓ All supplementary files ↗ Raw reads on ENA ↗

Also filed as BioProject PRJNA1416661 and SRA study SRP671499. Searching any of these in the dataset finder brings you back here.

NCBI GEO page ↗ Paper (PMID 42146372) ↗

Study design

3 × RPE1_Pol2ser5_R1T vs 3 × RPE1_Pol2ser5_R2T vs 2 × RPE1_H3K4me1_R1T vs 2 × RPE1_H3K4me1_R2T

Supports a between-group comparison across 10 samples.

4 replicated groups read from 10 sample titles; they account for 10 of them. Check it against the sample list below before relying on it.

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