GSM6597962: 293T_150DFO_POLII; Homo sapiens; ChIP-Seq
Cells were harvested and washed twice with ice-cold PBS and then fixed with paraformaldehyde (1% final) for 10 min at RT. Afterwards, the paraformaldehyde solution was quenched with 2.5 M (1/20) glycine, and then, cell pellets were washed twice with PBS. Sonication: The cell pellets were resuspended with lysis buffer 1 (50 mM HEPES, pH = 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, 1X protease inhibitors) and then incubated on nutator at 4 °C for 10 min. Afterwards, cell pellets were centrifuged at 500 g for 5 min and discarded supernatant. Then, cell pellets were washed with lysis buffer 2 (10 mM Tris-HCl, pH = 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 X protease inhibitors) and resuspended with lysis buffer 3 (10 mM Tris-HCl, pH = 8.0, 1 mM EDTA, 0.1% SDS, 1 X protease inhibitors). The final volume was adjusted to be 10 times the size of each cell pellet with lysis buffer 3. Sonication was performed with 1-ml Covaris tubes which were set to 10% duty factor, 175 peak intensity power, and 200 cycles per burst for 60–1200 s. Ten percent of 10X ChIP dilution buffer (10% Triton x-100, 1 M NaCl, 1% Na-Deoxycholate, 5% N-Lauroylsarcosine, 5 mM EGTA) was added to the lysate, and samples were centrifuged at maximum speed for 15 min at 4 °C to pellet debris. Immunoprecipitation: Antibody was added (~ 10 μg per purified antibody or 40 μl of anti-sera) to each sample. After incubation at 4 °C on nutator overnight, 100 μl Protein A/G Agarose beads were added for each sample for 2 h. The agarose beads were washed 4 times with RIPA buffer (50 mM HEPES, pH = 7.5, 500 mM LiCl, 1 mM EDTA, 1.0% NP-40, 0.7% Na-Deoxycholate), followed by once with ice-cold TE buffer (with 50 mM NaCl). After removing the residual buffer, the DNA for each IP sample was eluted with elution buffer (50 mM Tris-HCl, pH = 8.0, 10 mM EDTA, 1.0% SDS) and reverse cross-linked at 65 °C oven for 6–15 h, followed by protease K digestion at 55 °C for 2 h. The genomic DNA fragments were then further purified with Qiagen DNA purification kit (Cat. No. 28104) ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250-450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit.