GSM2151468: MB468.FOXR2_rep1; Homo sapiens; ChIP-Seq
Cells were rinsed with cold PBS, harvested in 200 μl of SDS lysis buffer supplemented with protease inhibitor mixture (Roche Applied Science) and 1 mM phenylmethylsulfonylfluoride (PMSF) per 1 × 106 cells, and sonicated to shear the DNA to yield fragments of 500 bp or less. Samples were then diluted with 9 volumes of ChIP dilution buffer before being precleared for 1 hour with 40 μl of a mixture of protein A/G agarose and salmon sperm DNA. Approximately 5 μg of FOXR2, MYC or MAX antibody or mouse normal immunoglobulin (IgG) were added to the precleared supernatant and incubated for 1 hour at 4°C. Forty μl of protein A/G agarose were added, and the mixture was incubated overnight at 4°C. Washes were sequentially performed with low-salt buffer, high-salt buffer, LiCl buffer, and twice with TE buffer. Immunoprecipitates were eluted in 500 μl of elution buffer (1% SDS, 0.1 M NaHCO3), followed by the addition of NaCl (20 μl). Crosslinking was reversed by overnight incubation of samples at 65°C and treatment with protease K (Sigma) for 2 hours at 45°C. The recovered DNA was extracted with phenol-chloroform and precipitated with ethanol. Quantification was performed by real-time PCR with the My IQ real-time PCR detection system and an IQ SYBR Green Supermix (Bio-Rad). Control IgG and input DNA values were used to normalize values from ChIP recovery. Library was contracted by the MD Anderson DNA sequencing Core facility following standard protocol.
GEO Accession
GSM2151468