GSM7506770: NIPBL Untreated +estradiol 72hrs_Input; Mus musculus; ChIP-Seq
Cells were fixed with 1% formaldehyde for 10 minutes at room temperature. The reaction was quenched with glycine to a final concentration of 125mM for 5 minutes at room temperature. ChIP was performed using ChIP-IT High Sensitivity kit (Active motif) according to the manufacturer's instructions. Briefly, cells were resuspended in chromatin preparation buffer and incubated on ice for 10 minutes. Nuclei were collected by centrifuging at 1000g for 5 minutes at 4 °C. The nuclear pellets were resuspended in ChIP Buffer and lysates were fragmented using Covaris E220 with the following settings: peak power -140W, duty factor - 5%, 200 cycles per burst, temperature=4°C for 900 seconds. Chromatin immune precipitation was carried out using 4µg of anti-RAD21 antibody (Abcam, #ab992). Protein G agarose beads were used for pulldown protein-DNA complexes. ChIP-Seq library construction was performed using Truseq ChIP Library Preparation Kit (IP-202-1012) according to the manufacturer's instructions.