GSM5322389: K562_Nailseq_EB_BrdU_rep2_HU041_LY105b; Homo sapiens; OTHER
Cells were harvested, washed with PBS, and applied to genomic DNA (gDNA) extraction with Proteinase K digestion as described previously.  Purified genomic DNA was subjected to Click reaction with 100 µM Biotin-PEG-azide, 2 mM CuSO4, 4 mM THPTA, and 10 mM Sodium ascorbate at 25℃ for 1 hour, followed by DNA precipitation. The copper in the click reaction hydrolyzes gDNA and fragments it into small fragments (around 200~400 bp in length), resulting in that each fragmented nascent DNA has little chance to contain both EdU and BrdU, simultaneously. Dissolved DNA was denatured at 95℃ for 5 min and phosphorylated by T4 PNK at 37℃ for 30 min.  For EdU- and BrdU-labeled samples, the denatured and modified DNA were purified by anti-BrdU (BU1/75) and Protein G beads for BrdU-labelled proportion, and the supernatants were denatured again and then incubated with Streptavidin C1 beads (Dynabeads) to isolate EdU-labeled proportion.  Isolated EdU- or BrdU-labeled ssDNA were ligated on-beads with two types of bridge adapters (Bridge adapter -1 and -2, see the resource table for sequence details), simultaneously, at room temperature for at least 4 hours as follow: 1 µM Bridge adapters each, 1,600 U T4 DNA ligase and 15% PEG-8000. After that, ligated DNA was harshly washed and tagged with Illumina P5-I5 and P7-I7 sequences as described (Yin J, et al., 2019, Cell Discov.).All the libraries were subjected to 2 × 150 bp Hiseq sequencing.