Multi-Contact 4C (MC-4C): long molecule sequencing of complex proximity ligation products to uncover local cooperative and competitive chromatin topologies v1

We present the protocol and data analysis toolbox for Multi-Contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites. Conventional chromosome conformation capture (3C) methods fragment proximity ligation products for efficient analysis of pairwise DNA contacts. In contrast, MC-4C is designed to preserve and collect large concatemers of proximity ligated fragments for long molecule sequencing on Oxford Nanopore or Pacific Biosciences platforms, thus allowing study of multi-way chromatin interactions. By inverse PCR with primers specific for a fragment of interest (the viewpoint) and DNA size selection, sequencing is selectively targeted to thousands of different complex interactions containing this viewpoint. A tailored statistical analysis toolbox employing data-intrinsic background models then discerns whether contacts between more than two regulatory sequences are mutually exclusive or, conversely, simultaneously happening at chromatin hubs. The entire procedure can be completed in two weeks and requires access to a third generation sequencing platform.