title: GSM1399546: Mmus E14-WT ChIP Differentiation Suz12 72h  rep. 1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were cross-linked with 1% formaldehyde in PBS for 10 min at room temperature and the reaction stopped with addition of glycine to a final concentration of 0.125M. Cells were harvested in SDS buffer (50 mM Tris at pH 8.1, 0.5% SDS, 100 mM NaCl, 5 mM EDTA). Pelleted cells were resuspended in IP buffer (100 mM Tris at pH 8.6, 0.3% SDS, 1.7% Triton X-100, and 5 mM EDTA) and sonicated to produce DNA fragments <1000 bp with an average size around 300 bp using a Diagenode Bioruptor. Lysates were precleared with protein A sepharose beads (GE Healthcare). Cleared samples (0.5 mg of chromatin) were immunoprecipitated O/N in IP buffer with the indicated antibodies: anti-Suz12 (D39F6 XP Cell Signaling), anti-Ezh2 (D2C9 XP Cell Signaling), anti-H3K27me3 (C36B11 Cell Signaling), anti-histone H3 (made in our lab, peptide purified by standard methods), anti-RNAPII N-20 (Santa Cruz, sc-899), IgG from rabbit (Sigma). Protein A beads were blocked O/N in 1X TE with 0,2 mg/mL sonicated herring sperm (Sigma) and 0,5 mg/mL BSA (New England Biolabs) before incubation with antibody-chromatin complexes for 3h at 4°C. Washes were performed first 3 times with Mixed Micelle buffer (150 mM NaCl, 20 mM Tris-Cl, pH 8.1, 5 mM EDTA, pH 8.0, 5% w/v sucrose, 1% Triton X-100, 0.2% SDS, 0.02% NaN3), twice with Buffer 500 (0.1% (w/v) deoxycholic acid, 1 mM EDTA, 50 mM HEPES, pH 7.5, 500 mM NaCl, 1% (v/v) Triton X-100, 0.02% NaN3), twice with LiCl/detergent buffer (0.5% (w/v) deoxycholic acid (sodium salt), 1 mM EDTA, 250 mM LiCl, 0.5% (v/v) Igepal, 10 mM Tris-Cl, pH 8.0, 0.02% NaN3) and one time in 1X TE. Immune complexes were eluted and reverse cross-linked in 1% SDS, 0.1 M NaHCO3 O/N at 65°C. DNA was recovered and purified using Qiagen PCR purification kit. ChIP was performed as described above with 1 mg of chromatin except that Protein A beads for precipitation were not blocked with BSA and herring sperm. The immunoprecipitated DNA was quantified by real-time qPCR and Qubit fluorometer (Life Technologies). DNA library for Illumina sequencing was prepared using the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina (New England Biolabs) using 10 ng DNA as input and following manufacturer’s instructions. Library cDNA was quantified and the quality assessed using the High Sensitivity DNA Kit with a 2100 Bioanalyzer (Agilent). Equimolar amounts of samples, with compatible indexes, were pooled for multiplex sequencing. Samples were sequenced at the National High-throughput DNA Sequencing Centre of the University of Copenhagen for 50 bp HiSeq Single Read sequencing using Illumina Sequencing Technology.
identifiers.namespace: GEO
identifiers.id: GSM1399546