GSM9305666: TCF3_Input_Knockout; Homo sapiens; ChIP-Seq
Crosslinked chromatin was fragmented into mono-nucleosomes through digestion with MNase. Briefly, isolated and crosslinked nuclei were MNase digested in 400μl MNase digestion buffer (0.3M Sucrose, 50mM Tris pH 7.5, 30mM KCl, 7.5mM NaCl, 4mM MgCl2, 1mM CaCl2, 0.125% NP-40, 0.25% NaDeoxycholate, supplemented with cOmplete™ Protease Inhibitor Cocktail (Roche)) with 100U MNase (Roche) at 37°C for 30 minutes. The digestion was then stopped with 5mM EDTA and the digested chromatin was solubilized in 1% SDS and eight pulses of 30 seconds sonication using a Bioruptor® Pico ultrasonic cell disruptor (Diagenode). 200μg of pre-cleared chromatin was immunopurified with incubation of 20μl of E2A antibody (CST #12258) over night. The next day, Dynabeads™ Protein A (Invitrogen, 10002D) were added and incubated for 4 hours at 4°C. The samples were subsequently washed, eluted and the DNA was purified by ethanol precipitation. NEBNext Ultra II