GSM2051869: A3 H3K9me3 ChIP-seq; 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 Kit.
GEO Accession
GSM2051869