GSM6430035: A375_PTEN_4C; Homo sapiens; OTHER
A375 cells were cross-linked by 2% formaldehyde, quenched with 0.125 glycine (Sigma-Aldrich, 50046), lysed with cold lysis buffer (50 mM Tris pH 7.5 (ThermoFisher, 15567027), 0.5% NP-40 (ThermoFisher, FNN0021), 150 mM NaCl (Sigma-Aldrich, S7653), 1% Triton X-100 (ThermoFisher, T8787), 5 mM EDTA (ThermoFisher, 15575020) and 1×protease inhibitors (ThermoFisher, 78425)), digested with NlaIII (NEB, R0125L) or HindIII (NEB, R0104) for 4 h at 37°C while shaking at 800 RPM for more than three times until the digestion efficiency reached over 90%, and then ligated with T4 DNA ligase (NEB, M0202) for overnight in a 7 ml reaction system after enzyme inactivation. The ligation sample was purified using Phenol/Chloroform/Isoamyl Alcohol (25:24:1) (ThermoFisher, 15593-049) before reversing the cross-links by adding Protein K (ThermoFisher, AM2548) for 6 h at 65°C and RNase A (ThermoFisher, 15596-018) for 1 h at 37°C. Next, the DNA samples were digested with DpnII (NEB, R0543L) or NlaIII (NEB, R0125) and ligated with T4 DNA ligase for more than 6 h in a 14 ml reaction system. The ligation product was purified using Phenol/Chloroform/Isoamyl Alcohol (25:24:1) and further purified using the QIAquick PCR Purification Kit (QIAGEN, 28106). The purified product was amplified by PCR using a high-fidelity DNA polymerase (Vazyme, N616-02) with 8 × 200 ng DNA. 4C-seq library was constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 according to the manufacturer's description, and sequenced on a HiSeq X Ten Platform.