title: GSM5351466: TSC_Repliseq_late_rep1; Mus musculus; OTHER
library_construction_protocol: We followed the BrdU-immunoprecipitation (IP)-based protocol, as described previously (Ryba et al., 2011). Cells were labeled by BrdU (Sigma #B5002) for 2 hours then collected by trypsinization. Cell pellets were resuspended by 5% FBS/PBS and EtOH was added to 75% final concentration for fixation. The ethanol fixed cells were sorted into early- and late-S-phase fractions (20,000 cells per each fraction) by FACS using Sony SH800 cell sorter as described previously (Takahashi et al., 2019). Genomic DNA was extracted from each fraction and fragmented by sonication. Then immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences #555627) was performed to enrich BrdU-labeled replicating DNA. After BrdU-IP, immunoprecipitated DNA samples were subjected to whole-genome amplification (WGA) followed by library preparation according to the previous study (Miura et al., 2020)