title: GSM1404657: ACH2 H3K9ac; Homo sapiens; ChIP-Seq
library_construction_protocol: 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). Pre-  cleared Dynabeads Protein A (100.02D, Life Technologies, Carlsbad, CA) were  bound with antibodies specific to the histone H3, tri methylated at K4  (rabbit polyclonal, ab8580, Abcam, Cambridge, UK) and acetylated at K9  (rabbit polyclonal, ab4441, Abcam, Cambridge, UK). Digested chromatins were  incubated with bead-bound antibody for overnight 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).
attributes.tag: GEO Accession
attributes.value: GSM1404657