GSM2461327: Dex 3h pre, 0h LPS RelA ChIP; Mus musculus; ChIP-Seq
For RelA ChIP, BMDMs were subject to crosslinking with 1% formaldehyde for 10 minutes at room temperature. For GR ChIP, we used a dual crosslinking method (Uhlenhaut et al., 2013) to detect both tethered and directly bound chromatin. BMDMs were subject to crosslinking with 2mM disuccinimidyl glutarate (DSG) for 30 min followed by 1% formaldehyde for 10 min at room temperature. After crosslinking, chromatin was isolated from about 4 × 10^7 cells, which allows 2 to 3 different immunoprecipitations per sample at a time, and then processed as follows. The lysis buffer to shear the chromatin contained 0.5% SDS, 10 mM EDTA (pH 8), 50 mM tris-HCl (pH 8), and proteinase inhibitor cocktail. Sonication was performed to shear the chromatin to generate DNA fragments with a size range of 400 to 500 base pairs. The sheared chromatin samples were diluted 1:5 in dilution buffer (0.01% SDS, 1.1% Triton-X, 1.2 mM EDTA (pH 8), 20 mM tris-HCl (pH 8), 167 mM NaCl, and proteinase inhibitor cocktail). For each ChIP, 200 µg chromatin DNA was precipitated with antibody coated bead complexes (Dynabeads Protein A for RelA ChIP; anti-IgG paramagnetic beads for GR ChIP, Invitrogen). sequencing libraries were generated using Illumina TruSeq V3 protocol and subject to 51 bp single-end sequencing on Illumina HiSeq2000.
GEO Accession
GSM2461327