GSM5322415: mESC_Nailseq_EH_Pol2_HU059_LY138c; Mus musculus; 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/HU-labeled samples, T4 PNK-treated DNA was incubated with C1 beads alone. All of the enrichment steps were performed at room temperature for 4 hours.  Isolated EdU-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.