Protocol for quantifying chromatin DNA replication by multiplex labeling and single-molecule localization microscopy

We present a protocol for imaging and quantifying chromatin DNA replication-associated signals in synchronized mammalian cells. We describe steps for cell-cycle enrichment, nucleoside analog-based metabolic labeling, and fluorescence detection. We then detail the procedure for dual-color STORM imaging and SR-Tesseler-based cluster analysis. It can be adapted to distinct questions in DNA replication biology by modifying synchronization schemes, pulse durations, labeling strategies, fluorescence detection channels, or analysis parameters. For complete details on the use and execution of this protocol, please refer to Zhang et al.¹.