title: GSM6132866: Hs578_PRRX1_ChIP_Input_cond1; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells (1 × 10^7) were fixed with 1% paraformaldehyde for 10 min at room temperature. DSG was then removed and replaced with fixing buffer (50 mM HEPES-NaOH (pH 7.5), 100 mM NaCl, 1mM EDTA) containing 1% paraformaldehyde (Electron Microscopy Sciences, 15714) and cross-linked for 10 min at 37 °C. Crosslinking was quenched by adding glycine to a final concentration of 0.125 M. The cells were washed with ice-cold PBS, harvested in PBS. The nuclear fraction was extracted by first resuspending the pellet in 1 ml of lysis buffer (50 mM HEPES-NaOH (pH 8.0), 140 mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, and 0.25% Triton X-100) for 10 min at 4 °C. Cells were pelleted and washed in 1 ml of wash buffer (10 mM Tris-HCL (pH 8.0), 200 mM NaCl, 1 mM EDTA) for 10 min at 4 °C. Cells were then pelleted and resuspended in 1 ml of shearing buffer (10 mM Tris-HCl (pH 8), 1 mM EDTA, 0.1% SDS) and sonicated in a Covaris sonicator. Lysate was centrifuged for 5 min at 14,000 rpm to purify the debris. Then 100 µl of 10% Triton X-100 and 30 µl of 5M NaCl were added. The sample was then incubated with 20 µl of Dynabeads Protein G (LifeTechnologies,10003D) for 1 h at 4 °C. Primary antibodies were added to each tube, and immunoprecipitation (IP) was conducted overnight at 4 °C. Cross-linked complexes were precipitated with Dynabeads Protein G for 2 hr at 4 °C. The beads were then washed in low salt wash buffer (20 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM EDTA, and 1% SDS) for 5 min at 4 °C, high salt wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS) for 5 min at 4 °C and LiCl wash buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS) for 5 min at 4 °C. DNA was eluted in elution buffer (100 mM sodium bicarbonate and 1% SDS). Cross-links were reversed overnight at 65 °C. RNA and protein were digested with 0.2 mg ml−1 RNase A for 30 min at 37 °C followed by 0.2 mg ml−1 Proteinase K for 1 h at 55 °C. DNA was purified with phenol-chloroform extraction and isopropanol precipitation. ChIP-seq libraries were prepared using the Rubicon ThruPLEX DNA-seq Kit from 1 ng of purified ChIP DNA or input DNA according to the manufacturer's protocol.