GSM4878374: ChIP-Seq_U2OS_RUVBL2_rep2; Homo sapiens; ChIP-Seq
the crosslinked cells were digested using MNase (200 U/ 20 million cells in 1 ml sonication buffer, for 15 min at 37 °C), then dissociated in Bioruptor Sonication Device (high energy for 30s on and 60s off, work on 20 cycles). Lysates were centrifuged twice at 1,2000 rpm for 10 min at 4 °C, 20 μl supernatant were sampled as input, the rest supernatant were transferred into 2 new DNase free tube (500 μl/ tube) , then 1 μl GFP abs (Abcam, no. ab290), and equal isotype IgG control (Millipore, no. 12-370, 12-371), or 1 μg of RPB1 abs (Abcam, No. ab817), INO80 abs (Proteintech, no.18810-1-AP), p400 abs (Bethyl, no. A300-541A), Smc1a abs (Bethyl, no. A300-055A), and 2 μl CTCF abs (ABclonal, no. A1133) or 5 μl RUVBL2 abs (ABclonal, no. A1905) were added for each ChIP reaction, respectively, and incubated overnight at 4 °C with low speed rotation, the antibody enriched DNA was extracted by Tris saturated equilibrium phenol (25:24:1) (pH8.0). The RUVBL1/2, Pol II (RPB1), CTCF and SMC1 ChIPed DNA were delivered to library preparation through TELP method (Peng et al., 2015), and mESCs INO80 and P400 ChIPed DNA and human and cancer cells RUVBL2 ChIPed DNA were subjected for library construction using the NEBNext Ultra II DNA library prep kit according to the manufacturer-supplied protocol, the 200~400 bps length amplified DNA were recovered and analyzed with Qubit assays and Fragment Analyzer, and finally subject to HiSeq Xten PE150 sequencing (Novogene, Beijing).