Protocol for identifying genomic binding sites of mitotic bookmarkers in Drosophila neural stem cells and cultured mammalian cells.

Mitotic bookmarkers label key gene loci on highly condensed chromosomes to preserve cell fate memory across mitosis. Here, we present a protocol for identifying binding sites of mitotic bookmarkers (BISMIBs) in developing tissues and cultured cells. We describe a workflow for isolating mitotic and interphase cells from Drosophila larval brains and HEK293T cells independent of cell-cycle synchronization. We further detail procedures for low-input in vivo CUT&Tag (Cleavage Under Targets & Tagmentation) sequencing. This protocol is applicable for identifying binding sites for mitotically retained factors in developing tissues. For complete details on the use and execution of this protocol, please refer to Shen et al.(1).