GSM5851210: scRepli Site-B deletion mutant 21; Mus musculus; OTHER
BrdU-IP Repli-seq samples: Cells were labeled with 50 µM BrdU for 2 h before harvest. The harvested 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 flowcytometer (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 with a SeqPlex kit (Sigma. SEQXE). Amplified gDNA was used to construct NGS libraries. Single-cell Repli-seq samples: Growing cells were fixed in 75% ethanol and single mid-S-phase cells were sorted with a Sony SH800 cell sorter in single-cell mode based on DNA content (PI intensity). Single cells were sorted directly into a 96-well plate with 6 µl of cell lysis buffer (447 µl of H2O, 3 µl of Proteinase K (Sigma, P4850), 50 µl of 10X single-cell lysis and fragmentation buffer (Sigma, L1043)), incubated at 55 °C for 1 h and then at 99 °C for 4 min for gDNA isolation and fragmentation. Then, 6 µl of the gDNA solution was subjected to WGA with a SeqPlex kit (Sigma, SEQXE). Amplified gDNA was used to construct NGS libraries. NGS libraries were constructed with a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs), according to the manufacturers' instructions.