GSM9178558: P3_unenrichment; Homo sapiens; OTHER
30 μg genomic DNA extracted from NCM460 or HCT116 cell lines was fragmented to about 250 bp by sonication, and then mixed with 100 mM of Azide-PEG3-Amino (2 μL) and 25 mM of K2IrBr6 (2 μL) in NaPi buffer (100 mM, pH 8.0) in a 50 μL reaction system for 30 min at 37 °C. The reaction was terminated by adding 300 μL of ice-ethanol, and then purified using MolPure®PCR Purification Kit (Yesean) to obtain the azide-modified cytogenomic DNA. Next, the obtained azide-modified cell genomic DNA was mixed with 0.01 mg of DBCO-IgG in a 100 μL reaction system at 37 ℃ for 2 h. After the reaction was completed, the IgG-coupled DNA product to be enriched was purified by magnetic bead purification and further enriched using protein G magnetic beads (New England Biolabs). In the enrichment process, 10 μL of magnetic beads were washed three times with 500 μL of binding buffer (0.1 M sodium phosphate buffer, pH 8.0). Then 1 μg of fragmented λDNA genome was added and pre-incubated for 1 h. Later, the DNA to be enriched was added to the magnetic beads and incubated at 37 ℃ for 30 min. After that, the beads were washed three times with 500 μL of binding buffer. Next, 50 μL of enzyme-free water was added to the beads for resuspension, and 1 μL of Proteinase K (40 mg/mL) was added and incubated at 55 oC for 1 h. The supernatant was aspirated and purified by the MolPure®PCR Purification Kit. The enriched nucleic acid product was subjected to end repair, adding A-tails, adapter ligation and PCR library amplification in accordance with conventional methods, and then the prepared cell genome libraries were used for subsequent sequencing analysis.
GEO
GSM9178558_r1