GSM7764486: Ube2iF/F SUMO1_GFP_Replicate 1; Mus musculus; ChIP-Seq
After one wash with ice cold PBS, 5x107 cells were resuspended farnham lysis buffer (5mM PIPES pH 8.0, 85mM KCl, 0.5% NP-40 and protease inhibitor). Nuclei pellets were collected by centrifugation at 2,000 rpm at 4℃ for 5 min, and resuspended in RIPA buffer  for sonication using sonic dismembrator at 4℃ with the following settings: 35% amplitude in 10-minute rounds of on/off for 30s for 4 cycles. The sonicated chromatin was centrifuged at 13,000 rpm for 15 min at 4℃. 50 ul of sonicated chromatin was prepared as input chromatin DNA. The rest of supernatant was added into antibody-coupled beads and then rotated together at 4℃ overnight. The next day, beads were washed 5 times with RIPA buffer  for 4 mins, 5 times with LiCl wash buffer (100mM Tris-HCl pH 7.5, 500mM LiCl, 1% NP-40, 1%SDS), and once with TE buffer. Immunoprecipitated chromatin was eluted in 200 ul IP elution buffer (1% SDS, 0.1m NaHCO3) by incubation at 65℃ for 1 hour and centrifuged at 13,000 rpm for 5 min at room temperature. Supernatant containing ChIP DNA was reverse crosslinked by 65℃ incubation overnight. 20 ul of proteinase K and 8 ul of RNase A were added to digest protein and RNA, then purified by using QIAquick PCR purification kit according to the manufacturer's instructions.