Genome wide glucocorticoid receptor (GR) binding sites upon cortisol treatment in human CD8 T cells
Genome wide glucocorticoid receptor (GR) binding sites upon cortisol treatment in human CD8 T cells
Primary human CD8⁺ T cells were obtained from healthy adult donors (with appropriate consent/ethics). CD8⁺ T cells were purified by immunomagnetic negative selection, activated in vitro with plate-bound anti-CD3 and anti-CD28 antibodies, expanded in IL-2, and re-stimulated with anti-CD3/anti-CD28 in the presence of hydrocortisone (cortisol; 100 nM) or vehicle control for 48 h prior to chromatin immunoprecipitation. Chromatin immunoprecipitation (ChIP) was performed using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology). Cells were crosslinked with 1% formaldehyde for 10 min at room temperature, quenched with 1× glycine for 5 min, and washed with ice-cold PBS. Nuclei were isolated and chromatin was digested with micrococcal nuclease (0.3 µL in 200 µL ChIP buffer) for 20 min at 37 °C, followed by sonication (Branson 450-D; 36% amplitude; four 5-s pulses; on ice). GR was immunoprecipitated overnight at 4 °C using two anti-GR antibodies (PA1-511A and D6H2L) and immune complexes were captured using ChIP-grade Protein G magnetic beads (2 h, 4 °C). After low- and high-salt washes, chromatin was eluted (1× elution buffer, 30 min, 65 °C), crosslinks were reversed (65 °C, 2 h) in the presence of NaCl and Proteinase K, and DNA was purified for downstream library preparation and sequencing. Purified GR ChIP DNA and matched input DNA were submitted to Novogene (UK) for DNA sample QC and ChIP-seq library preparation using standard Illumina-compatible workflows (end repair/A-tailing, adapter ligation, PCR amplification, and size selection). Final libraries underwent provider QC before sequencing.