GSM8067217
In summary, the fixed cells underwent three times washing with 0.1% BSA/PBS. The cells were then resuspended in 80 µl of Antibody Buffer (a mixture of 4 µl 0.5 M EDTA and 1 ml 0.01% Digitonin-Wash Buffer (Dig-Wash Buffer), supplemented with cocktail, 10 mM sodium butyrate, and 0.05% Triton X-100) that contained 1.6 µg of the primary antibody. This mixture was incubated at 4°C for 4 hours on a rotator with 16 rpm. The cells were subsequently washed twice with 180 µl of Dig-Wash Buffer. The secondary antibody (used at a 1:500 ratio) was incubated under the same conditions, except for a shorter duration of 10 minutes. After two times washing with Dig-Wash Buffer, the cells were suspended in 80 µl of Dig-Wash Buffer (containing 0.05% Triton-X, cocktail, and 10 mM sodium butyrate) that also contained 0.83 µM PAT-MEA/B. This was done at 4°C for 1 hour. The free PAT-MEA/B were then dislodged with 180 µl Dig-Wash Buffer containing 0.05% Triton X-100 three times at 5 min each time. The reaction was activated by suspending the cells with 10 µl Reaction Buffer (10 mM TAPS-NaOH pH 8.3, 5 mM MgCl2, 10% DMF, 300 mM NaCl, 0.05% Triton-X, 0.01% Digtonin and supplemented with cocktail, 10 mM sodium butyrate), followed by gently flicking and incubating at 37°C for 1 h in an Eppendorf ThermoMixer with 300 rpm. The reaction was gently mixed once after 30 min incubation and was halted by adding 10 µl of 40 mM EDTA, followed by further incubation at 4°C for 10 min. The samples were then washed with 0.1% BSA/PBS and finally resuspended in 5 µl of Lysis Buffer (0.1 mg/ml Proteinase K, 10 mM Tris-HCl pH 8.5, and 0.05% SDS), and lysed at 55°C for 3 hours in a PCR cycler. Finally, 1.8% Triton X-100 with 5 mM PMSF was added to the cell lysate and incubated at room temperature for 10 minutes to inactivate the proteinase K. This was followed by a 5-minute incubation at 55°C to quench the SDS in the reaction. After PCR enrichment using 2x KAPA master mix,fragments larger than 1 kb were first eliminated using 0.4x AMPure beads, after which the library was purified using the Zymo DNA Clean and Concentrator-5 Kit.