GSM9378610: Human3_TAID_rep1; Homo sapiens; OTHER
For each sample, 10 µL of Concanavalin A (ConA) beads were washed with 100 µL Wash Buffer, resuspended in 10 µL Binding Buffer, and incubated with 200,000–300,000 cells for 10 min at room temperature (RT). After magnetic separation, the primary antibody diluted in Antibody Buffer was added and incubated overnight at 4 °C. The beads were then washed, incubated with secondary antibody for 1 hours at RT, and washed three times with Dig-Wash Buffer. Subsequently, pA–Tn5 Buffer was added, and samples were incubated for 1 hour at RT, followed by three washes with Dig-400 Buffer. Tagmentation was performed with 40 µL of Tagmentation Buffer at 37 °C for 1 hour. The reaction was stopped with EDTA, SDS, and Proteinase K, followed by incubation at 55 °C for 1 hour and 75 °C for 15 min. DNA was purified using Speed Beads, washed twice with 80% ethanol, and eluted in pre-heated dd-water containing 0.1% Tween-20. A 10 µL aliquot of eluted DNA was extended with 15 µL 2× Equinox PCR mix, 0.375 µL Bst 3.0 DNA polymerase (8 U/µL), and 5 µL nuclease-free water, incubated at 68 °C for 30 min to fill adapter gaps, followed by 95 °C for 5 min to inactivate the enzyme. Index 5 and Index 7 primers were then added, and PCR amplification was performed as described (Supplementary Table 11.3). The final library was purified using DNA Clean beads (1.2× sample volume) for downstream applications.