title: GSM8744858: Scramble-KD MEF TNF ChIP-seq (replicate 1); Mus musculus; ChIP-Seq
library_construction_protocol: After stimulating cells, cells were washed with PBS and double-crosslinked with 100 mM disuccinimidyl glutarate/PBS solution (DSG, ProteoChem) for 30-min at room temperature followed by 1% methanol-free formaldehyde/PBS solution (Fisher). Crosslinking was quenched with addition of 125 mM glycine for 5-min at room temperature. Cells were washed once with PBS and harvested by scraping. Cell nuclei was isolated and resuspended in 1 mL RIPA buffer with protease inhibitors, then DNA was sonicated using a Model 120 sonic dismembrator (Fisher) to obtain DNA fragments in a general range of 200-1000 bp. DNA fragments were incubated with 1uL anti-RelA antibody (Diagenode) bound to 30uL protein G magnetic beads (NEB). Beads were washed and immunoprecipitated DNA was eluted and decrosslinked with incubation at 65C overnight. Eluted DNA was PCR amplified (Roche) using ready mix primers (Roche). Amplified DNA was then purified with Ampure XP beads and quantified by dsDNA Qubit (ThermoFisher). Libraries were pooled and size selected with PippinHT (Sage Science) before 50 bp paired-end sequencing.