title: GSM8791227: Yjlab_chip_S015_female_rep1_k27ac; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were then fixed with 1% formadelhyde for 10min at room temperature and then quenched with glycine for 5min. Crosslinked cells were resuspended in 1ml of pre-chilled cell lysis buffer supplemented with 1:500 protease inhibitor and 1:100 PMSF. Cells were incubated on ice for 20min. After cell lysis, nuclei were pelleted at 7200rpm for 2min at 4℃. Nuclei were then resuspended in 1ml nuclear lysis buffer supplemented with 1:500 protease inhibitor and 1:100 PMSF for 20min on ice. Nuclei lysate (chromatin) was then subject to sonication on a Qsonica Q800R3 sonicator (80% amplitude, 20s ON/40s OFF for 17min). Sonicated samples were centrifuged at 15000g for 10min at 4℃ to remove cell debris. Chromatin in the supernatant was collected and diluted with 4 volumes of IP dilution buffer (20mM Tris-HCl pH=8.0, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS) containing 1:500 protease inhibitor and 1:100 PMSF. 50ul of A/G agarose beads (Santa Cruz, sc-2003) was then added into the system to for preclear. Preclear of chromatin was performed at 4℃ for 8h with rotation. Precleared chromatin was then incubated with 35ml A/G agarose beads (A:G = 1:1) pre-bound with ChIP grade antibody mentioned in article at 4℃ overnight. Beads were washed once with IP wash buffer I (20mM Tris-HCl pH=8.0, 2mM EDTA, 50mM NaCl, 1% Triton X-100, 0.1% SDS), twice with high salt buffer (20mM Tris-HCl pH=8.0, 2mM EDTA, 500mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP wash buffer II (10mM Tris-HCl pH=8.0, 1mM EDTA, 0.25M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate) and twice with TE buffer (10mM Tris-HCl pH=8.0, 1mM EDTA pH=8). All washing steps were performed on ice. Beads were moved to room temperature and DNA was eluted in 200µl fresh Elution Buffer (100mM NaHCO3, 1%SDS). ChIP chromatin was reverse crosslinked at 65℃ overnight in the presence of proteinase K. Samples were supplemented with 10ml of 3M sodium acetate (pH=5.2), and DNA was purified with QIAquick PCR purification kit (QIAGEN, 28106). ChIP-seq libraries were constructed using the AHTS Universal DNA Library Prep Kit for MGI (Vazyme, NDM607-02) according to the manufacture's protocol.