GSM5173347: CPT 7h_BrdUIP_LateS_replicate 1; Homo sapiens; OTHER
Cells were washed twice with ice-cold PBS, trypsinized, and fixed in 75% ethanol. Fixed cells were resuspended in PBS contained 1% FBS, stained with propidium iodide (50 µg/ml) for 15 min in the presence of RNase A (100 µg/ml). These cells were sorted into early and late S phase fractions by SH800 flow cytometer (SONY). After phenol-chloroform extraction of DNA from 10,000 cells, sonication was performed to cut the DNA into an average size of about 100-700 bp. Then, immunoprecipitation with anti-BrdU mouse monoclonal antibody (BD Biosciences, #555627) was performed in each fraction to enrich BrdU-substituted replicating DNA. Isolated early and late replicating DNA was amplified individually by WGA (Sigma. SEQXE). NGS libraries were constructed from early- and late-replicating DNA after WGA with a TruSeq DNA PCR-Free High Throughput Library Prep Kit (Illumina, San Diego, CA, USA) and NEXTflex® DNA-Seq Kit, both according to the manufacturers' instructions.