GSM6754374: H3K27ac_d39; Homo sapiens; ChIP-Seq
DNA was extracted using AMPure XP beads (Beckman Coulter Genomics, A63881). Chip sample libraries were prepared according to Illumina Genomic DNA protocol as described (Mikkelsen et al., 2007). Chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) is a method used to analyze protein interactions with DNA. ChIP-seq combines with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. 106 of differentiated cells from Day 26, Day 39, Day 55 stages were cross-linked using 1% formaldehyde. The lysate with sodium dodecyl sulfate-based reagents, and chromatin was sonicated for 18 cycles of (60 sec On, 60 sec Off)  using Bioruptor 208.The sonicated samples were immunoprecipitated using magnetic beads 25ul protein A (Invitrogen cat.10002D) and 25ul protein G (Invitrogen cat.10004D). The samples were reverse crosslinked using Proteinase K overnight at 650C. The sonicated fragments were 300-500bp size. The DNA fragments were purified using phenol-chloroform protocol209. ChIP was performed using antibodies against H3K27ac (Abcam Ab4729) and H3K27me3 (Abcam Ab4729). To verify the quality of experiment, ChIP samples were taken to qPCR with primers for positive and negative controls (table 6).  Prepared libraries from ChIP and input DNA were sequenced using HiSeq instrument (Illumina, USA). the ChIP libraries were analyzed and mapped to hg19 using BWA 210 and peaks were called using MACS211.