GSM2838638: IgG_rn5; Rattus norvegicus; ChIP-Seq
Lysates were clarified from sonicated nuclei and proteine-DNA complexes were isolated with antibody. Schwann cells (~20 million cells) were cross-linked in 1% formaldehyde at room temperature for 10 min and then quenched with 125 mM glycine for 5 min. Cells were rinsed, resuspended in lysis buffer (50 mM HEPES, pH 7.6, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.25% Triton X-100 and 0.5% NP-40 and protease inhibitor cocktail) and incubated on ice for 10 min. Lysates were centrifuged and pellets were resuspended in nuclei washing buffer  (200 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM Tris pH 8.0, and protease inhibitor cocktail) with gentle rocking at 4°C. Nuclei were pelleted and resuspended in 1 ml of sonication buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 0.5 mM EGTA and protease inhibitor cocktail). Nuclear suspensions were sonicated with a Covaris S220 sonicator (total time 8 min). Antibody (chromatin: antibody = 4:1) was added to chromatin and incubated at 4°C overnight. Chromatin-protein complex was immunoprecipitated with protein A/G plus agarose beads and washed sequentially, twice with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.0], 150 mM NaCl), twice with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.0], 500 mM NaCl) and twice with LiCl buffer (0.25 M LiCl, 1% IGEPAL CA630, 1% deoxycholic acid, 1 mM EDTA, 10 mM Tris [pH 8.0]) and once with 1xTE buffer. Immunoprecipitates were then eluted in elution buffer at 65°C for 1 h. Eluted chromatin was subjected to reverse-crosslink and chromatin fragment purification with QIAGEN PCR purification columns. ChIP-Seq libraries were prepared using NEBNext ChIP-Seq Library Prep Kit according to manufacturer's instructions.
GEO Accession
GSM2838638