title: GSM9381630: K562 GATA1 bulk D&D-seq secondary Ab; Homo sapiens; ATAC-seq
library_construction_protocol: For all the following centrifugations, cells were centrifuged in a swing-bucket centrifuge at 300g for 5 minutes before fixation and 600g for 5 minutes after fixation. About 200K to 1 million cells were collected and resuspended in 400 µl PBS. Then, 16% methanol-free formaldehyde (Thermo Fisher Scientific, PI28906) was added for fixation (final concentration 0.1%) at room temperature for 5 minutes. Glycine (final concentration 125 mM) was added to stop the cross-linking, followed by a wash with 1 ml of PBS. Fixed cells were permeabilized in 200 µl permeabilization buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, 1% BSA, 1× protease inhibitors) on ice (7 minutes for cell lines, 5 minutes for primary cells), followed by a wash with 200 µl cold wash buffer (20 mM HEPES pH 7.6, 150 mM NaCl, 0.5 mM spermidine, 1% BSA, 1× protease inhibitors). After cell counting, 100K-400K cells were transferred to PCR tubes and resuspended in 100 µl antibody buffer (20 mM HEPES pH 7.6, 150 mM NaCl, 2 mM EDTA, 0.5 mM spermidine, 1% BSA, 1× protease inhibitors), with 1 µl (1:100 dilution) of the antibodies. The cells were incubated at 4 °C with slow rotation overnight. For experiments with secondary antibodies, the cells were washed with antibody buffer and resuspended in 100 ul antibody buffer with 1 µl (1:100 dilution) of the secondary antibodies and incubated at room temperature for 1 hour. The cells were washed once with 200 ul wash buffer, and another time with 200 ul D&D binding buffer. The cells were resuspended in 50 ul D&D activation buffer with 100 uM DddA_NT, and incubated at room temperature on a rotator for 1 hour. The cells were then washed with 200 ul D&D binding buffer, resuspended in 50 ul DnD activation buffer, and incubated at 37 °C for 1 hour for deamination. After D&D activation, the cells were washed once with 200 ul 1x Tris-TD buffer and resuspended in 50 ul 1x Tris-TD buffer with loaded TnY or Tn5 (volume depends on its  concentration and needs to be titrated to get the optimal tagmentation). To initiate tagmentation, the reaction was incubated at 37°C for 1 hour. To extract DNA, 1 ul 10% SDS, 2 ul proteinase K (NEB, P8107S), and 3 ul 0.5M EDTA were added to the reactions, which were then incubated at 55°C for 1 hour, followed by column-based DNA purification per the manufacturer's instruction (Zymo, D5205). For library preparation PCR, we used uracil-tolerant DNA polymerase (NEBNext Q5U Master Mix, NEB M0597S) and Nextera-compatible indexing primers to amplify the purified DNA. To increase the efficiency of the initial gap fill-in step, we spiked in 1 ul of non-hot-start polymerase Bst 3.0 (NEB, M0374). The PCR steps are: 72°C for 5 min; 98°C for 30 s; 12 cycles of 98°C for 10 s, 55°C for 30 s and 72 °C for 30 s; followed by 72 °C for 5 min. Bulk samples are sequenced PE150 or PE100 for about 40M reads per sample.