GSM7951531: G3M,CON,P300_SI_rep1; Homo sapiens; ChIP-Seq
Cells were resuspended with 80 µl Antibody Buffer (mix 4 µl 0.5 M EDTA with 1 ml 0.01% Digitonin-Wash Buffer (Dig-Wash Buffer) supplemented with cocktail, 10 mM sodium butyrate and 0.05% Triton X-100) containing 0.4 µg primary antibody. The mixture was incubated at 4°C for 4 h in a rotator with 30 rpm. Cells were then washed twice with 180 µL Dig-Wash Buffer. The secondary antibody (1:500 use) was incubated in the same buffer and condition except for the duration of 10 min. After washing with Dig-Wash Buffer twice, cells were suspended with 80 µl Dig-Wash Buffer (with 0.05% Triton-X, cocktail, and 10 mM sodium butyrate) containing 0.83 µM PAT-MEA/B at 4°C for 1 h. 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 the Eppendorf ThermoMixer with 300 rpm. The reaction was gently mixed once after 30 min incubation and stopped by the addition of 10 µl 40 mM EDTA followed by further incubation at 4°C for 10 min. Samples were washed with 0.1% BSA/PBS and finally resuspended in 5 µl Lysis Buffer and lysed at 55°C for 3 h in a PCR cycler. 1.8% Triton X-100 with 5 mM PMSF was added to the cell lysate and incubated at RT for 10 min to quench the proteinase K followed by incubation at 55°C for 5 min to quench SDS in the reaction. For H3K27ac and P300 samples, Spike-In DNA (0.1 mg/ml Proteinase K, 10 mM Tris-HCl PPH 8.5 and 0.05% SDS, 20 pg Tn5 tagmenated Drosophila DNA) was added in the Lysis Buffer. PCR amplification was performed by the addition of 1μl of 10 μM i5 index primer (5'-AATGATACGGCGACCACCGAGATCTACAC[8 bp index]TCGTCGGCAGCGTC-3'), 1 μl of 10 μM i7 index primer (5'-CAAGCAGAAGACGGCATACGAGAT[8 bp index] GTCTCGTGGGCTCGG-3'), 30 μL of 2ⅹKAPA master mix and 23 μl of 1 mM MgCl2 to the cell lysate. The reaction was set up by incubation at 72 °C for 5 min, 98°C for 45 s, 16-18 cycles of amplification (98°C for 15 s, 63°C for 30 s, 72°C for 1 min), and final 72°C extension for 5 min. After PCR, for histone-ChIP, DNA fragments were size-selected: first 0.4X AMPure beads to remove > 1 kb fragments, and second 0.6X AMPure bead to retain 200-1000 bp fragments; for TF-ChIP, > 1 kb fragments were first removed by using 0.4X AMPure beads, then library was purification by Zymo DNA Clean and Concentrator-5 Kit. All libraries were sequenced with paired-end 150-bp reads on NovaSeq 6000 platform (Illumina).