Illumina HiSeq 1500 sequencing; Single-cell replication timing analysis of hTERT-RPE1 cells depleted for SAF-A protein
Single-cell replication timing analysis of hTERT-RPE1 cells depleted for SAF-A protein
Cells were fixed in 70& ethanol and followed by propidium iodide staining.  Single G1 and mid-S phase cells were collected by FACS, cells were sorted with a Sony SH800 cell sorter using the single-cell mode.  hTERT-RPE1 cells were grown in DMEM supplemented with 10% FBS. Cells were treated with either control siRNA (Dharmacon D-001100-01) or SAF-A siRNA (Dharmacon L-013501-00-0005) for 24 hr 3 days before collecting cells. Only the 1st read of the paid-end sequencing was used.  Single-cell data (FACS sorted samples): sequence reads were trimmed to remove adapter sequences using the cutadapt program before mapping. We performed a two-step adapter trimming, first removing the Illumina adapter based on the index of each NGS library and then removing the SEQXE adapter. As the SEQXE adapter sequence was not available, we empirically estimated it as the sequence that repeatedly appeared near the 5' end. Single-cell data (samples collected by the micromanupitator): sequence reads were trimmed to remove the WGA4 universal sequences using seqtk program (\"seqtk trimfq -b 30 -e 30\", trimming 30bp of both 5' and 3' ends) before mapping. Genomic DNA samples were prepared as described (Takahashi et al. Nat Genet. 2019. 51: 529-540; Miura et al. Nat Protoc. 2020. 15: 4058-4100). 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).  Extracted gDNA samples were subjected to WGA with a WGA4 kit (Sigma, WGA4). NGS libraries were constructed with a NGS LTP Library Preparation Kit (KAPA, KK8232) according to the manufacturer's instructions, and as described (Takahashi et al. Nat Genet. 2019. 51: 529-540; Miura et al. Nat Protoc. 2020. 15: 4058-4100) with TruSeq adapter.