A low-input Micro-C protocol for high-resolution 3D genome mapping

Standard Micro‑C protocols typically require millions of cells, limiting their application to rare cell populations. Here, we present an optimized low‑input Micro‑C workflow that requires only 100,000 cells. By downsampling both our low‑input dataset and a control dataset from 5 million cells to 120 million raw read pairs, we demonstrate that all key architectural features—compartments, topologically associating domains (TADs), and chromatin loops—are reliably detected from as few as 100,000 cells. The low‑input protocol achieved a high cis interaction ratio (96.1%) and low PCR duplication rate (3.0%), indicating high library complexity and low background noise. Applying this method to investigate acute CTCF degradation, we observed the loss of loops and TAD boundaries in CTCF‑degraded samples, consistent with previous reports. Our optimized protocol enables nucleosome‑resolution 3D genome mapping for sample‑limited studies.