GSM1931783: BJ1 ZFP57; Mus musculus; ChIP-Seq
ChIP was performed on formaldehyde cross-linked chromatin isolated from cells grown on 10 cm dishes to ~80% confluency. Briefly, the cells were detached by adding 0.05% trypsin at 37 °C for 3 min. Formaldehyde was added to the cells resuspended in PBS at a final concentration of 1% and the cells were incubated at room temperature for 15 min with shaking. The reaction was stopped by addition of glycine to a final concentration of 0.125 M. Approximately 3x107 cells were washed twice in ice-cold PBS, centrifuged and resuspended in lysis buffer 1 (50 mM HEPES pH 8, 10 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40 and 0.25% Triton X-100) for 90 min at 4 °C. Isolated nuclei were lysed in lysis buffer 2 (10 mM Tris–HCl pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) for 60 minat 4 °C. The chromatin was sheared in sonication buffer (10 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% N-lauroylsarcosine) to an average size of 100–400 bp using the S220 Focused-ultrasonicators (Covaris). For each IP, 100 μg of sonicated chromatin were diluted in a final volume of 600 μl with sonication buffer and pre-cleared with 30 µl protein A/G agarose beads (SantaCruz) for 4 h at 4 °C on a rotating wheel. Anti-ZFP57 antibody (8 μg, Abcam ab45341), anti-KAP1 antibody (7 μg, Abcam ab10483) and anti-Histone H3K9me3 (7 μg, Abcam 8898) or rabbit IgG were added to the pre-cleared chromatin and incubated overnight at 4 °C on a rotating wheel. Chromatin was precipitated with 30 μl protein A/G agarose beads for 4 h at 4 °C with rotation. The beads were then washed five times with 500 μl RIPA buffer  (10 mM Tris–HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 1% Triton X-100and 0.1 % SDS) and once with each of the following buffers: WASH buffer (50 mM HEPES, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 500 mM NaCl and 0.2% NaN3), LiCl buffer  (0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris pH 8) and TE buffer (10 mM Tris pH 8, 1 mM EDTA). The bound chromatin was eluted in 100 μl TE buffer. Crosslinks were reversed by incubation O/Nat 65°C after addition of 1 μl RNAse cocktail (Ambion) and 2h at 50°C after addition of 2.5μl SDS 20% + 2.5μl 20mg/ml proteinase K (Sigma). The DNA was extracted by using the QIAquick Gel Extraction Kit (Qiagen). Immunoprecipitated or 1% input DNAswere analysed by real-time PCR using SBYRGreen PCR Master Mix (Bio-Rad) on a C1000 Thermal Cycler (Bio-Rad). Two nanograms of DNA from immunoprecipitated and input chromatin were used for Illumina library preparation. Libraries were generated by using the NuGen Ovation Ultralow Library System v2 Kit.
GEO Accession
GSM1931783