GSM7109960: K562_Input_ChIP-seq; Homo sapiens; ChIP-Seq
[ChIP-seq] Cells were crosslinked with 1% formaldehyde (vol/vol, Thermo) for 10 min at room temperature, followed by quenching with 125 mM glycine for 5 min. Crosslinked chromatin was then fragmented using a sonicator (Scientz) for 28 cycles (30 seconds on, 30 seconds off). [CUT&Tag] Cleavage under targets and tagmentation (CUT&Tag) libraries were prepared using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (TD901, Vazyme Biotech). 100,000 cells were used for each assay. These cells were bound to conA beads and incubated with anti-H3K27ac (ab4729, Abcam) or anti-NIPBL antibody (A301-779A, Bethyl) for 2h at room temperature. After washing, a secondary antibody (Goat Anti-Rabbit IgG, Sangon) was diluted (1:100) in DIG Wash buffer and incubated with cells at room temperature for 1h on a shaker with slow rotation. The nuclei were then incubated with the Hyperactive pG-Tn5 Transposon for 1h at room temperature with slow rotation. After washing three times with Dig-300 buffer, these cells were then resuspended in tagmentation buffer and incubated for 1h at 37 °C. Chromatin was purified using standard phenol/chloroform/alcohol extraction method. Fragmented DNA were then amplificated by PCR and then purified by magnetic beads. The final libraries were sequenced on an Illumina HiSeq 2500 System. [ChIP-seq] The sonicated lysate was incubated with a H3K27ac antibody (ab4729, Abcam). Dynabeads Protein G beads (Invitrogen) were added to the extract–antibody complex. After DNA purification, ChIP-Seq library was prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645, NEB) following the manufacturer's protocol and sequenced on an Illumina HiSeq 2500 System.