GSM9700813: DEK_CUT-Tag_MOLM13_shDEK_1; Homo sapiens; OTHER
The CUT&Tag assay was performed using NovoNGS CUT&Tag 3.0 High-Sensitivity Kit (Novoprotein) according to the manufacturer's protocol. 5,000-10,000 cells were harvested and resuspended in 45 µL wash buffer. Then 6 µL ConA beads were added to the sample and incubated at 37°C for 10 min. Then the supernatant was removed by magnet adsorption 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, 1× PIC). Then, 0.5 µL antibody was added to the sample and incubated at 4°C overnight. The supernatant was removed by magnet adsorption and the beads were resuspended in 50 µL secondary antibody buffer, containing 0.5 µL goat anti-rabbit IgG antibody. After incubation at 37°C for 60 min, the beads were resuspended in 50 µL ChiTag Buffer, containing 0.5 µL ChiTag transposome. After incubation at 37°C for 60 min, the beads were resuspended in 50 µL tagmentation buffer. Then, 1 µL 10% SDS was added to the sample and incubated at 55°C for 10 min, and DNA was extracted using 2×DNA clean beads (Vazyme) and amplified with i5 and i7 primers (Vazyme) by 17-20 PCR cycles. The libraries were purified by 1.2×DNA clean beads (Vazyme).