GSM7846932: MSC, KDM4D, DFO treatment; Homo sapiens; OTHER
The DNA fragments were extracted using phenol chloroform. The CUT&Tag assay was performed using NovoNGS® CUT&Tag 2.0 High-Sensitivity Kit (for Illumina®) (Novoprotein Scientific Inc., Cat# N259-YH01-01A) according to the manufacturer's instructions. Briefly, cells were harvested with trypsin and enriched by ConA-magnetic beads. A total of 50,000 cells were re-suspended and washed twice with 100 μL Dig-wash Buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 1× protease inhibitor cocktail; 0.05% digitonin). The samples were incubated with primary BRD4 antibody (1:100, 4°C, 18 h) and secondary antibody (1:200, 25°C, 1 h). After incubation, the beads were washed three times in Dig-Hisalt Buffer (0.05% digitonin, 20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, 1× protease inhibitor cocktail). Cells were incubated with the protein A-Tn5 transposome at 25°C for 1 h and washed three times in Dig-Hisalt buffer to remove unbound protein A-Tn5. The cells were re-suspended in 50 μL Tagmentation buffer (10 mM MgCl2 in Dig-Hisalt Buffer) and incubated at 37°C for 1 h. The tagmentation was terminated with the addition of 1 μL of 10% SDS at 55°C for 10 min. Cleavage under targets and tagmentation (CUT&Tag)