GSM3895246: RelA-DB Input DNA, rep 2; Mus musculus; ChIP-Seq
After stimulating MEFs with TNF, MEF cells were washed with PBS and double cross-linked with 100mM disuccinimidyl glutarate/PBS solution (DSG, ThermoFisher Scientific) for 30-min followed by 1% methanol-free Formaldehye/PBS solution (ThermoFisher Scientific) for 15-min. Cross-linked cell pellets were quenched with 125mM Glycine for 5-min. Cell lysates were washed with PBS, incubated in lysis buffer, and sonicated to obtain DNA fragments of 250 to 1000 bp. Cells were sonicated with Diagenode Bioruptor 300 sonication system. DNA fragments were incubated with 5 ug anti-RelA antibody (Santa Cruz Biotechnology, sc-372) overnight and immunoprecipitated DNA fragments were subjected to reverse cross-linking and cleaned up by AMPure XP magnetic beads. 5ng of input or ChIP DNA were subjected for library synthesis without size selection using NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs) according to manufacturer's instruction. ChIP DNA libraries were quantified using Qubit 2.0 Fluorometer.