MTBP Cut&Run of FACS sorted cell cycle populations in HCT116 cells

The selection of replication origins plays a crucial role in eukaryotic DNA replication. However, the mechanisms of origin selection in humans are not yet well-defined. Recent work in yeast has shown that the recruitment of Sld3-Sld7 to origins during G1 is involved in origin selection. In humans, TICRR and MTBP are the orthologs of Sld3 and Sld7, respectively. In this study, we have mapped the genomic binding locations of MTBP in G1, early S, late S, and G2 populations of cells. Overall design: We utilized HCT 116 cell lines with all MTBP alleles tagged with mClover in addition to expressing Fucci(CA) fluorescent cell cycle reporters. Cells were FACS sorted based on Fucci fluorescence into G1 (mCherry), S (AmCyan), or G2 (double positive) populations. Cut&Run experiments were performed with an anti-GFP antibody that specifically binds to the mClover tag. To obtain background signal measurements, we simultaneously conducted Cut&Run experiments on the untagged cell lines. Subsequently, the generated Cut&Run reads were aligned to the genome and used to call peaks.