OEX002081
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NAIL-seq_EdU_HU |
Genomic |
Cells with indicated treatment were harvested, washed with PBS, and applied to genomic DNA extraction with Proteinase K digestion as described previously (Yin et al., 2019). Purified genomic DNA was subjected to Click reaction with 100 µM Biotin-Azide, 2 mM CuSO4, 4 mM THPTA, and 10 mM Sodium ascorbate at 25℃ for 1 hour, followed by DNA precipitation. Dissolved DNA was denatured at 95℃ for 5 min and phosphorylated by T4 PNK at 37℃ for 30 min.
For EdU- and/(or) BrdU-labelled samples, the denatured and modified DNA were purified by anti-BrdU (BU1/75) and Protein G beads for BrdU-labeled proportion, and the supernatants after anti-BrdU-ChIP were denatured again and then incubated with Streptavidin C1 beads (Dynabeads) to isolate EdU-labeled proportion. For EdU/HU labeled samples, T4 PNK-treated DNA were incubated with C1 beads directly. All the enrichment was incubated at room temperature for 4 hours.
Enriched EdU- or BrdU-labeled ssDNA were ligated on-beads with two types of bridge adapters (Bridge adapter-1 and -2, see key resource table for sequence details) 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 were harshly washed and tagged with Illumina P5-I5 and P7-I7 sequence as described (Yin et al., 2019). All the libraries were subjected to 2 × 150 bp Hiseq sequencing. |
2019-11-05 |