title: GSM3537250: ChIP-Seq: ARID1A-WT1 H3K27Ac rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: The chromatin immunoprecipitation (ChIP) assay was performed as previously described on the two independent clones of human endometrial epithelial ARID1AWT and ARID1AKO cells (PMID: 26953344) with the following modifications. Cells were grown in 15 cm dishes, and approximately 1.2×107 cells were cross-linked using Diagenode ChIP cross-link Gold and 1% formaldehyde according to the manufacturer's instruction. Nuclear contents were extracted using the truChIP Chromatin Shearing kit (Covaris) according to the manufacturer's instructions. Chromatin was sheared for 12 mins in shearing buffer by using a Covaris E220 focused ultrasonicator. The fragment size was ensured to be between 200-600 bp. The sonicated lysates were diluted 5-fold with ChIP dilution buffer (0.1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 1× protease inhibitor), and immunoprecipitated with rotation overnight at 4°C with 0.5-3 µg of antibodies. The antibody/chromatin complex was then precipitated for 3 h by Protein A/G DYNAL magnetic beads (40 µl of 1:1 mixture). Antibody-protein complexes bound to beads were washed once with low salt buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 1% Triton-X100, and 2 mM EDTA), once with high salt buffer (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 0.1% SDS, 1% Triton-X100, and 2 mM EDTA), once with LiCl buffer (250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, and 10 mM Tris-HCl pH 8.0), and twice with TE, pH 8.0. For sequential ChIP, immunocomplex from the first ChIP was eluted with 10 mmol/L dithiothreitol in Tris-EDTA buffer at 37°C and subjected to second round of ChIP. DNA and protein complexes were digested in TE buffer containing 1% SDS, 200 mM NaCl and 1U Proteinase K (Thermo Scientific) at 56°C for 2h, and cross-linking was reversed by heating at 65°C for 4h. DNA fragments were purified using a QIAquick PCR Purification Kit in 55 µl of EB elution buffer. Tru-seq ChIP-seq library preparation and sequencing using a NextSeq500 platform with single-end reads of 75 bases were performed by JHMI Deep Sequencing and Microarray Core
identifiers.namespace: GEO
identifiers.id: GSM3537250