GSM1847879: gDNA_CD4_H3K27ac_Graves_rep3; Homo sapiens; ChIP-Seq
Peripheral blood mononuclear cells were extracted using Ficoll-Paque (GE Healthcare) gradient centrifugation. CD8+ T-cells and CD4+ T cells were purified by consecutive positive separation using microbeads (CD4+ #130-045-101; CD8+ #130-045-201) and AutoMACS technology (Miltenyi Biotec) according to the manufacturer's protocol. The purified cells were fixed in 1% formaldehyde and stored as cell pellets in a -80°C freezer. ChIP assays were performed using Auto Histone ChIP-seq kit (Diagenode) with minor modifications. Briefly, IP-Buffer (2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, 1% Triton X-100) was used in exchange for kit Buffer H in IP reactions. DNA from 4-6 x 106 cells was fragmented with the Bioruptor sonicator (Diagenode) to fragments of 200-400 basepairs. Sheared chromatin from 1 million cells was immunoprecipitated with 2 μg of anti-H3K4me3 antibody (Millipore 07-473) or 2 μg of anti-H3K27Ac antibody (Abcam Ab4729) using the SX-8G IP-Star Automated System (Diagenode). ChIP-DNA was purified with MinElute PCR Purification Kit (Qiagen). The purified ChIP and input DNAs were quantified by Qubit 2.0 Fluorometer (Life Technologies) prior to library preparation with the Ovation® Ultralow DR Multiplex System 1-96 kit (NuGeneration Limited) according to manufacturer’s instructions. The barcoded libraries were quantified by Qubit 2.0 Fluorometer and validated with Agilent 2200 TapeStation analysis (Agilent Technologies, Inc.). The ChIP-seq libraries were sequenced with HiSeq 2500 (Illumina) producing 50 bp paired-end reads. Phage PhiX sequencing library was included as 1% spike-in in the sequencing runs.
GEO
GSM1847879