GSM8733385: GR.Dex1h.2; Homo sapiens; ChIP-Seq
Cells were wash with ice-cold PBS (2x), Fixed in FA 1.5% for 10 min. Tris-Glycine buffer was added for 1 minute and cells were washed ice cold PBS (2x). Cells were lysed and sonicated (Diagenode bioruptor) in order to get DNA fragments of 300-800pb. IP was performed overnight using anti-GR or ER antibodies, in IP buffer, ytRNA and from 15 ug of chromatin quantified by Qubit. Protein-A coated magnetic beads were added the next day for 4 h. Pulled-down DNA was washed multiple times, extracted in SDS based buffer and incubated at 65°C for reverse crosslinking. DNA was treated with RNase A and proteinase K, and purified using Zymo ChIP DNA Clean & Concentrator kit. DNA concentration was assessed using Qubit. NEBNext Ultra II DNA #E7645S kit