Protocol for the generation of low-input Hi-C sequencing libraries of FACS-isolated mitotic cells

High-throughput chromosome conformation capture (Hi-C) is a powerful tool to investigate 3D genome architecture. Here, we present a protocol for preparing low-input Hi-C libraries from mitotic cells isolated by fluorescence-activated cell sorting (FACS) to study chromatin conformation in mitotic cells. We describe steps for mitotic arrest, harvest and fixation of cultured cells, staining with an anti-Mitotic Protein Marker (MPM2), and isolation of mitotic cells. We then detail procedures for quantifying input material for Hi-C in mitotic cells and library preparation of Hi-C ligated material. For complete details on the use and execution of this protocol, please refer to Nichols et al.¹.