Multiplex Chromatin Interactions with Single Molecule Precision. Multiplex Chromatin Interactions with Single Molecule Precision

Emerging 3D genome mapping efforts suggest complex chromosomal folding structures. However, the true multiplex nature of chromatin interactions has yet to be fully explored. Here, we describe a chromatin interaction analysis by droplet-based sequencing (ChIA-Drop). In ChIA-Drop, individual chromatin complexes are partitioned into droplets that contain a gel bead of DNA-barcoded primers, such that tethered chromatin DNA fragments are uniquely indexed and amplified for sequencing and mapping to demarcate multiplex chromatin contacts. Thus, ChIA-Drop can identify complex chromatin interactions with unprecedented single-molecule precision, which is not possible using methods that analyze pairwise contacts via proximity ligation. We demonstrate that multiplex chromatin interactions predominantly contribute to topologically associated domains with high heterogeneity, and that multivalent promoter-centered interactions provide a topological model for gene transcription. Overall design: The microfluidics device (10X Genomics’ Chromium instrument) produces Gel beads in Emulsion (GEM) with chromatin complexes for complex-specific DNA barcoding and amplification. Barcoded DNA amplicons from all GEMs (droplets) are pooled for DNA sequencing and mapping for identifying chromatin interactions.