GSM7847040: CR_TAL1_HDP2_Day4; Homo sapiens; OTHER
Two million HUDEP-2 cells were collected and subjected to a single wash with PBS. Subsequently, the cell pellets were resuspended in 300 μl of NE buffer (comprising 20mM HEPES-KOH, pH 7.9, 10mM KCl, 0.5mM spermidine, 0.1% triton X-100, 20% glycerol, and 1x protease inhibitor) and incubated on ice for a duration of 20 minutes. ConA beads, measuring 30 μl, were washed twice with 250 μl of cold binding buffer (consisting of 20mM HEPES-KOH, pH 7.9, 10mM KCl, 1mM CaCl2, and 1mM MnCl2), and subsequently resuspended in 30 μl of binding buffer. This mixture was then combined with the nuclear solution in NE buffer and incubated at room temperature for 10 minutes. The supernatant was removed, and the cells were resuspended in 250 μl of cold blocking buffer (composed of 20mM HEPES, pH 7.5, 150mM NaCl, 0.5mM spermidine, 0.1% BSA, 2mM EDTA, and 1x protease inhibitor), followed by a 5-minute incubation at room temperature. The cells were then washed with 500 μl of cold wash buffer (comprising 20mM HEPES, pH 7.5, 150mM NaCl, 0.5mM spermidine, 0.1% BSA, and 1x protease inhibitor), and subsequently resuspended in 150 μl of cold wash buffer containing the primary antibody at a dilution of 1:100. This suspension was incubated for 2 hours at temperature. Following the incubation with the primary antibody, the supernatant was removed, and the cells were washed twice with cold wash buffer. The cells were then resuspended in 250 μl of cold wash buffer containing pA-MNase at a dilution of 1:2000 (resulting in a final concentration of 600ng/ml) and incubated at 4℃ for 1 hour with rolling. After the incubation, the supernatant was removed, and the cells were washed twice with cold wash buffer before being resuspended in 150 μl of cold wash buffer. The sample was subsequently cooled on ice for 10 minutes. To the sample, 3 μl of 100mM CaCl2 was added and mixed rapidly, followed by an incubation on ice for 30 minutes. The reactivation process was halted by the addition of 150 μl of 2x stop buffer (consisting of 200mM NaCl, 20mM EDTA, 4mM EGTA, 50ug/mL RnaseA, and 40ug/mL glycogen). The samples were then centrifuged for 5 minutes at 16000g, and the supernatant was collected. To this supernatant, 3 μl of 10% SDS and 2.5 μl of 20mg/ml proteinase K were added, followed by an overnight incubation at 50℃. The DNA was subsequently purified using phenol-chloroform extraction and ethanol precipitation. NEBNext Ultra II DNA library Prep Kit (E7645S) was employed to construct the library as described.