title: GSM2151471: MB468.MAX_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: 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.
attributes.tag: GEO Accession
attributes.value: GSM2151471