title: Illumina HiSeq 2000 sequencing; EZH2-mediated H3K27me3 targets transcriptional circuits of neuronal differentiation ChIP-seq
design: EZH2-mediated H3K27me3 targets transcriptional circuits of neuronal differentiation ChIP-seq
library_construction_protocol: Cells at the various time-points (72 hours or 10 days after Cre induction) were cross-linked in 1% formaldehyde for 10 min and the crossliking was stopped by incubating in 0.125 M glycine for 5 min. After washing cells were harvested in SDS buffer (0.5% SDS, 50mM Tris-Cl pH 8.1, 100mM NaCl, 5mM EDTA pH 8, 0.02% NaN3).  Prior to sonication, the lysate was diluted 2:1with dilution buffer (5% Triton X-100, 100mM Tris-Cl pH 8.6, 100mM NaCl, 5mM EDTA pH 8.5, 0.02% NaN3). Sonication was performed with Covaris sonicator generating DNA fragments with a bulk size around 250bp. Chromatin was quantified with Lowry method. After sonication, 600μg of chromatin and 12μg of anti-H3K27me3 (#9733BF Cell Signalling) were used for immunoprecipitation overnight at 4°C and recovered the following day using Dynabeads Protein G (Life Technologies). 1% of chromatin prior to immunoprecipitation was used as input. Immunoprecipitate was washed three times in low salt and one time in high salt wash buffer (1% Triton-X, 150mM or 500mM NaCl, 20mM Tris-Cl pH 8.0, 0.1% SDS, 2mM EDTA pH 8.0). One additional wash was performed in TE. Elution and crosslink reverse was performed under standard condition. DNA was then purified using PCR purification kit (QIAGEN).  Libraries were prepared by the sequencing facility at IEO according to the protocol described by Blecher-Gonen and colleagues (PMID: 23429716)