GSM3374250: WT ESC BrdUIP_LateS replicate 2; Mus musculus; OTHER
Cells were washed twice with ice-cold PBS, trypsinized, and then fixed in 75% ethanol. These cells were resuspended in PBS containing 1% FBS, stained with propidium iodide (20 μg/ml) for 30 min in the presence of RNase A (250 μg/ml), and then were sorted into early and late S phase fractions by FACSCalibur flow cytometer (BD Biosciences). After phenol-chloroform extraction of DNA from 10,000 cells, sonication was performed to cut the DNA into an average size of about 100-600 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). Libraries were constructed using Ion Plus Core Module for AB Library Builder System (Thermo Fisher Scientific, #4477683) with Ion Xpress Barcode Adapters 1-16 Kit (Thermo Fisher Scientific, #4477683) according to the standard instruction.
GEO Accession
GSM3374250