GSM7890101: Glucocorticoid Receptor, IL4, 24 hours, rep2, batch 2; Mus musculus; ChIP-Seq
Macrophages were crosslinked with 1% methanol-free formaldehyde (Thermo) for 10 min (H3K27ac) or double-crosslinked with 2 mM disuccinimidyl glutamate (Proteochem, c1104) (GR) for 30 min followed by 1% methanol-free formaldehyde for 10 min at room temperature (GR). Nuclear extracts were prepared  and chromatin DNA was sonicated using Bioruptor Pico (Diagenode, 8 cycles, 30s on/off bursts) to generate DNA fragments ~130-600 bp. (200-700 bp – reconcile). After sonication, 10% of nuclear extract was saved as input and rest incubated with protein A/G agarose (Santa Cruz) for 30 min at 4 °C. Precleared extracts were incubated with 40 μL of 50% protein A/G agarose and 5 g of normal rabbit IgG (Santa Cruz Biotech), H3K27ac (Active Motif, 39133), GR (Thermo Fisher Scientific, PA1-511A) at 4 °C overnight. The following day, beads were washed 8x with modified RIPA buffer containing 500 mM LiCl and 2x with TE + 50 mM NaCl buffer. DNA was extracted after reversing crosslinks and purified with Qiagen PCR purification kit. Resulting DNA was analyzed by qPCR (primer sequences are listed in Supplementary Data 1) with 28S as an internal control and occupancies expressed relative to the normal IgG baseline. Libraries were prepared using NEBNext Ultra II Library Prep Kit for Illumina (NEB), size selected (150-500 bp), and PCR-amplified for 15 cycles