Name: GSM7181571
Instrument: Illumina NovaSeq 6000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Collected the cultured cells in centrifuge tube and then washed with 1X PBS buffer. After centrifugation and removing the supernatant, 1% formaldehyde in PBS buffer was used to cross-link DNA and proteins for about 10min at 37 ℃. After that, 0.125 M of glycine was used to stop the crosslinking reaction. the cells were washed with precooled PBS with 0.5% bovine serum and by PBS supplemented with protease inhibitor compound (PIC). The cells were collected by centrifugation at 850 rpm for 3 min after each wash. The cells were resuspended in 200 μl ice-cold lyses buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 7.5 plus PIC) and then thawed on ice for 10 min to allow cell lyses. The cell lysate was sonicated using Bioruptor Pico to generate chromatin fragments of size range from 100 to 800 bp. One-tenth of the sonicated chromatin sample was separated for input control. The remaining chromatin was immunoprecipitated in ChIP dilution buffer (1% Triton, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5) with 4 μg of antibody that has been pre-incubated with protein A/G magnetic beads (Invitrogen; 10003D). The immunoprecipitation reaction was incubated overnight at 4 °C and the beads were washed twice with each of the following buffers at 4 °C: RIPA buffer (10 mM Tris, 1 mM EDTA, 0.1% SDS, 0.1% Na-deoxycholate, 0.1% Triton X-100); RIPA buffer plus 0.3 M NaCl; LiCl buffer (0.25 M LiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and TE buffer. The captured DNA and input control sample were reverse cross-linked by eluting the beads with elution buffer (1% SDS, 0.1 M NaHCO3) at 65 °C for 2 h, respectively. The eluted DNA was purified by phenol-chloroform extraction and precipitated with ethanol and glycogen. The obtained DNA was subjected to library preparation and sequenced on Illumina sequencing platform.