GSM7987121: EK8,Healthy,H3K27ac; Homo sapiens; ChIP-Seq
30mL of peripheral blood was collected in EDTA tubes from patients with SLE (n=11) and psoriasis (n=15) and from age- and sex-matched healthy donors (n=11 and n=15 respectively) from The University of Tartu, Estonia. PBMCs were obtained by Ficoll Paque (GE Healthcare) density gradient centrifugation. CD4+ T cells were collected by magnetic isolation using CD4 MicroBeads (Miltenyi) on the AutoMACs (Miltenyi) according to the manufacturer's instructions. 1 million isolated CD4+ T cells were cross-linked with an 11% formaldehyde buffer, lysed and subjected to sonication using a Bioruptor sonicator UCD-200 (Diagenode) to obtain chromatin fragments of 100–500 bp. Sheared chromatin was immunoprecipitated with 2 μL of anti-H3K27ac antibody (Abcam, ab4729) and 2 μL of Dynabeads M-280 magnetic beads (Sheep Anti-Mouse IgG, Sheep Anti-Rabbit IgG, Invitrogen) using the SX-8G IP-Star Automated System (Diagenode). ChIP samples were decrosslinked at 65°C for 4 hours. After incubation, the samples were treated with 0.2 mg/mL RNaseA (RNase Cocktail Enzyme Mix, Ambion, Life Technologies) and 0.4 mg/mL Proteinase K (Thermo Scientific). DNA was purified with the MinElute PCR Purification Kit (Qiagen) according to the manufacturer's protocol. ChIP-Seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina according to manufacturer's instructions. Library quality and concentration were determined using an Agilent Bioanalyzer DNA1000 chip. Libraries were sequenced on an Illumina NextSeq 500 Sequencer (75-bp single-end mode).