GSM1622306: ER_ChIPSeq; Mus musculus; ChIP-Seq
2 × 10e7 live cells were resuspended in SBSS and cross-linked with 1% formaldehyde for 5 min at 37°C. Cross-linking was quenched by addition of glycine to 125 mM and incubating for 5 min at room temperature. Cross-linked cells were sonicated on ice for twenty 10-sec bursts, cooling on ice for 15 sec between bursts. A total of 2 × 10e6 cell equivalents were used per ChIP. Lysates were incubated with the appropriate antibodies overnight at 4°C. Protein A or G Dynabeads (Invitrogen) were added to samples for 2 h,followed by washing with low-salt (150 mM NaCl), high-salt (500 mM NaCl), and LiCl salt buffers, and finally with TE. The complexes were then eluted overnight at 65°C in the presence of 1 mg/ml RNAse A (Sigma-Aldrich). The following day, the samples were incubated with 10 mg/ml proteinase K (Sigma-Aldrich) at 55 °C overnight. Following phenol:chloroform and chloroform extractions, DNA was precipitated using 1 mg/ml glycogen (Roche) and 95% ethanol overnight at −20°C. DNA fragments were made blunt using the END-IT DNA repair kit (Epicentre) and a 3'-dA overhang added using Exo-Minus Klenow DNA Polymerase (Epicentre). Illumina paired-end (PE) adapters were added using the Fast-Link DNA Ligation kit (Epicentre), and the fragments were amplified twice using Illumina PE primers and PfuUltra II Fusion HS DNA polymerase (Stratagene). Each round of PCR was followed by gel purification and sizing of the fragments.
GEO Accession
GSM1622306