GSM6738698: U-937_CUT2; Homo sapiens; OTHER
Cells were harvested and washed twice in 1 mL wash buffer (20 mM HEPES PH 7.5, 150 mM Nacl, 0.5 mM Spermidine, 1x PIC (Bimake)), and then resuspended in 300 μL wash buffer. Concanavalin A coated beads were activated by washing twice in binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM MnCl2, 1 mM CaCl2). 10 μL activated beads were then added to the cell suspension. After 30 min's incubation at room temperature, the supernatant was removed and the beads were resuspended in 50 μL antibody buffer (20 mM HEPES pH 7.5,150 mM Nacl, 0.5 mM Spermidine, 0.0125% Digitonin, 2 mM EDTA, 0.1% BSA, 1x PIC). Then 0.5 μL antibody (FOXO3A) was added and incubated for 2 hours at room temperature. After the supernatant was gently removed on DynaMag (Thermo), the beads were resuspended in 100 μL Dig wash buffer (20 mM HEPES pH 7.5,150 mM Nacl, 0.5 mM Spermidine, 0.0125% Digitonin, 1x PIC) containing 1 μL Guinea Pig anti-Rabbit IgG antibody. After incubation for 1 hr at room temperature, the beads were then washed three times in 800 μL Dig wash buffer, followed by resuspended in 100 μL Dig 300 wash buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 0.0125% Digitonin, 1x PIC). Then 0.04 μM pA-Tn5 adapter complex (Vazyme S603) was added and incubated for 1 h at room temperature. Beas were washed three times in 800 μL Dig 300 wash buffer and resuspended in 300 μL tagmentation buffer (20 mM HEPES pH 7.5, 300 mM Nacl, 0.5 mM Spermidine, 0.0125% Digitonin, 10 mM MgCl2, 1x PIC) and incubated for 1 hr at 37°C. And then 10 µL of 0.5 M EDTA, 3 µL of 10% SDS and 1 µL of 20 mg/mL Proteinase K were added and incubated at 55°C for 1 hr to stop tagmentation.The DNA was extracted by PCI and amplified with i5 and i7 primers (Vazyme, TD202) by 16-18 cycles' PCR. DNA libraries were prepared for sequencing using standard Illumina protocols. The amplified products were purified by Ampure XP beads to remove >1 kb fragment and to enrich the 200-1000 bp fragments for high-throughput sequencing.