title: GSM2904950: RPE1_100cells_MidS_rep3; Homo sapiens; OTHER
library_construction_protocol: For BrdU-IP samples, we followed our routine protocol as described (Ryba et al., Nat. Protoc. 2011). A Sony SH800 cell sorter was used in the ultra-purity mode, fractionating early and late S-phase populations. The BrdU-IP protocol has been described in detail (Ryba et al., Nat. Protoc. 2011), except that we used a Bioruptor UCD-250 (Sonic Bio) for gDNA sonication in high output mode, with ON/OFF pulse times of 30 sec/30 sec for 6 minutes. After BrdU-IP, immunoprecipitated DNA samples were subject to WGA with a SeqPlex kit (Sigma, SEQXE).   For early S-phase population samples, 200,000 cells from the first half of S-phase were sorted by Sony SH800 cell sorter, and gDNA was isolated using a Qiagen kit (Qiagen #69504, DNeasy Blood & Tissue Kit) and fragmented to 200–300 bp with a Covaris ultrasonciator (model: S220, tube: microTUBE snap-cap) according to the manufacturer's instructions (peak incident power: 175, duty factor: 10%, cycles per burst: 200, treatment time: 120), followed by cleanup and size selection via Agencourt AMPure XP SPRI beads (Beckman Coulter).  For single or 100 cells samples, cells were sorted with a Sony SH800 cell sorter using the single-cell mode. Sample preparations were based on the previous study (Baslan et al., Genome Res. 2015). Extracted gDNA samples were subjected to WGA with a SeqPlex kit (Sigma, SEQXE). Amplified gDNA was purified and size-selected with Agencourt AMPure XP SPRI beads and the SEQXE adapter sequence was removed by the primer removal enzyme Eco57I (Sigma, SEQXE). The DNA fragment size peak should be within 150–200 bp, which was confirmed by a capillary electrophoresis system, MultiNA (Shimadzu). NGS libraries were constructed with a NGS LTP Library Preparation Kit (KAPA, KK8232) according to the manufacturer's instructions, with slight modifications based on the previous study (Kadota et al., Sci. Rep. 2017). For a multi-plex NGS run, a SeqCap adapter kit A/B (Roche, 07141530001/ 07141548001) and NEXTflex DNA barcode (Bio Scientific, NOVA) were used. The samples were subjected to NGS on an Illumina Hiseq 1500 system (80, 84, 127-bp length, single-read or paired-end read). For NGS on Ion Proton system, 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).