GSM1423145: iPSCp_H3K27me3; Mus musculus; ChIP-Seq
Cells were resuspended with digestion buffer (50mM Tris-HCL, pH7.6, 1mM CaCl2, 0.2% triton X-100) and 5mM butyrate, 0.5mM PMSF, x proteinase inhibitor. The chromatins were digested with Mnase and enzyme was inactivated by adding the stop buffer (10mM Tris, pH7.6, 5mM EDTA). The chromatin was immunoprecipitated with antibodies specific to the H3K4me3 (ab8580, Abcam, Cambridge, UK), and H3K27me3 (07-449, Millipore, Billerica, MA) in RIPA buffer (10 mM Tris, pH7.4, 1mM EDTA, 0.1% SDS, 0.1% Na-Deoxycholate, and 1% Triton X-100). Bead complex was captured by magnetic stand and washed sequentially twice with RIPA buffer, RIPA + 0.3 M NaCl, and LiCl buffer (0.25 M LiCl, 0.5% NP40, 0.5% Na- Deoxycholate) and once with 1X TE + 0.2% Triton X-100, and 1X TE. Proteinase K digestion and phenol/chloroform extraction was performed to isolate the DNA. Sequencing libraries were manually generated for high-throughput sequencing. Either end of DNA strands was filled using the Epicentre DNA END-Repair kit (Epicentre Biotechnologies, Madison, WI). Adenine was added at 3’ ends by  Taq DNA polymerase (New England Biolabs, Beverly, MA) under dATP conditions. Illumina’s adaptor oligomers was ligated at both ends using Quick Ligation kit (Qiagen, Germantown, MD). After purified twice, DNA libraries are amplified using Solexa primers (Illumina, San Diego, CA) and Phusion PCR master mix (Thermo Scientific, Hudson, NH).