GSM6690677: MCE2-rep4 (scRepli-seq); Mus musculus; OTHER
For E/L Repliseq experiments, cells were labeled for 2 h with 50 µM BrdU (Sigma-Aldrich) before being fixed with 70% ethanol. We used a Sony SH800 cell sorter in ultrapurity mode to fractionate the early- and late-S phase populations based on DNA content (PI intensity). We followed our routine BrdU-immunoprecipitation (IP)-based protocol except that we used a Bioruptor (Cosmo Bio, Tokyo, Japan) for gDNA sonication to obtain DNA fragments of 100~700 bp. After BrdU-IP, the purity of early and late S phase cell fractions was validated by PCR using specific primer sets that detect early and late replicating regions. The immunoprecipitated DNA samples were then subjected to whole genome amplification (WGA) using a SeqPlex kit (Sigma-Aldrich, SEQXE). Amplified early- and late-replicating DNA were used to construct NGS libraries. For single-cell Repli-seq experments, single mid-S-phase cells were sorted with a Sony SH800 cell sorter in single-cell mode based on DNA content (propidium iodide intensity). Single cells were sorted directly into a 96-well plate with 6 µl of cell lysis buffer [447 µl H2O, 3 µl Proteinase K (Sigma, P4850), 50 µl 10× single-cell lysis and fragmentation buffer (Sigma, L1043)], incubated at 55°C for 1 h and then at 99°C for 4 min for genomic DNA isolation and fragmentation. Then, 6 µl of the genomic DNA solution was subjected to WGA using a SeqPlex kit (Sigma, SEQXE). Amplified gDNA was used to construct NGS libraries. Next generation sequencing (NGS) libraries were constructed using an NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs), according to the manufacturer's instructions