GSM8252645: Control_DMSO2_D2; Homo sapiens; OTHER
Cells were fixed with 1% Formaldehyde For library construction, MTU87 or LTAU87 cells with or without belinostat treatment were cross-linked using 1% formaldehyde for 10 min at room temperature and quenched by using 125 mM glycine. Nuclei were isolated by incubating the cross-linked cells in cytosolic buffer (15 mM Tris-HCl pH 7.5, 5 mM MgCl2, 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, 300 mM sucrose, and 1% NP40) for 10 min on ice with occasional agitation. Nuclei were precipitated by spinning at 1000 × g, 4 °C for 5 min and the supernatant was discarded. Open chromatin DNA was labeled with biotin by incubating the nuclei in the presence of 2.5 U of Nt.CviPII (NEB R0626S), 10 U of DNA polymerase I (M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μL of 1× NEB buffer 2. The labeling reaction was carried out at 37 °C in a thermo-mixer at 800 RPM for 2 h. We added 20 μL of 0.5 M EDTA and 2 μg of RNase A to the labeling reaction and incubated it at 37 °C for 0.5 h to stop the reaction and digest RNA. Finally DNA–protein cross-linking was reversed by adding 20 μL of proteinase K (NEB, P8107S) and 20 μL of 20% SDS to the reaction and incubating at 65 °C for overnight. Biotin-labeled genomic DNA was extracted using phenol chloroform.                Biotin-labeled genomic DNA were sonicated into 150-bp fragments (Covaris) and 1 μg of DNA was end-repaired, dA-tailed, and ligated with NEB Illumina adaptor (E7370S). Without further purification, the ligation product was mixed with 50 μL of Streptavidin magnetic beads (Invitrogen 65001, blocked using 0.1% cold fish gelatin in 1× PBS overnight at 4 °C) in 1 mL of B&W buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl). Biotin-labeled open chromatin DNA was captured by streptavidin magnetic beads at 4 °C for 2 h with end-over-end rotation. The beads were washed four times with B&W buffer plus 0.05% of Triton X-100 followed by one wash with TE plus Triton X-100. The beads were resuspended in 40 μL of nuclease-free water and 4 μL was used for library amplification using PCR (NEB, E7370S). Ten PCR cycles were usually sufficient to generate the amount of library DNA needed for sequencing.