title: GSM1483903: MEF.H3K9me3.ChIP-Seq; Mus musculus; ChIP-Seq
library_construction_protocol: Mononucleosomes were prepared according to Cell Signaling SimpleChIP® Enzymatic Chromatin IP protocols. For MNase-ChIP, 18 μg nucleosomes were incubated with 10 μg antibodies for each immunoprecipitation overnight at 4°C. 30 μl protein G dynabeads were then added for 2 hr at 4°C with rotation. Immune complexes were washed with low salt wash buffer three times and high salt wash buffer once. Mononucleosomes were eluted twice from the protein G magnetic beads and reversecrosslinked with addition of 12 μl 5 M NaCl and 4 μl 20 mg/ml protease kinase (PK) and overnight incubation at 65°C. Then the ChIPed DNA was purified with MinElute PCR purification Kits (Qiagen) and quantitated by Quant-iTTM Picogreen® dsDNA reagent (Invitrogen). Regular ChIP-seq for H3, H3K4me3, H3K27e3, H3K9me3 and CTCF was performed as described in a previous study (Huang et al., 2013). For RNA-Seq, cultured cells were harvested and dissolved in Trizol for total RNA extraction and treated with DNase I (Ambion) to remove any potential contaminated DNA fraction. We generated ChIP-seq libraries by using the ChIP-seq kit from Illlumina. For nucleosome preparation, we excised the band between 220-370 bp. For the libraries from CTCF, we excised the bands between 300-500 bp. For RNA-Seq, the library generation and sequencing were conducted by Beijing Genome Institutes (BGI) in Shenzhen. ChIP-Seq for Samples 1-15, and RNA-Seq for Samples 16-18
attributes.tag: GEO Accession
attributes.value: GSM1483903