GSM3931690: HepG2 JARID2 2nd [ChIP-seq]; Homo sapiens; ChIP-Seq
The cells lysates was sonicated to generate DNA fragments of 200-500 bp, centrifuged for 10 min at 12,000 g, and the supernatant was directly used for immunoprecipitation. Place the tube containing the Pierce™ ChIP-grade Protein A/G Magnetic Beads on the magnetic, then, discard the supernatant. Wash the beads for three times with 1ml 0.1M sodium phosphate Buffer. Resuspend the beads with 200ul 0.1M sodium phosphate Buffer. Add 10 ug target antibody (CST, 5646s) and Rabbit IgG (CST, 2729s) antibody to the tube containing beads. Incubate mixture on a rotating wheel 2h at 4°C. Place the tube containing the beads on the magnetic stand for 1 minute. Then, discard the supernatant. Resuspend beads with the cells lysates. Rotate the sample for overnight at 4 °C. Place on the magnetic, then, discard the supernatant. The beads were sequentially washed with LiCl IP Wash Buffer (100 mM Tris (PH 7.5), 500 mM LiCl, 1% NP-40 and 0.5 deoxycholate) for five times, TE buffer (10Mm Tris (PH 8.0), 1mM EDTA (PH 8.0)) for one time, respectively. Resuspend sample with 100ul Elution Buffer (100 mM NaHCO3, 1% SDS) and reverse cross-linked by overnight incubation at 65 ℃. After sequential RNase A and proteinase K treatment, DNA fragments were purified by phenol extraction and ethanol precipitation. Resuspend the RNA pellet in10ul water. To generate libraries with the ThruPLEX® DNA-seq Kit (R400427, Rubicon Genomics) according the manufacturer's procedure. Briefly, purified DNA fragments were end-repaired, adenylated, ligated to adaptors and PCR amplification. Used the ThruPLEX® DNA-seq Kit. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina NextSeq 500 system for 100 nt pair-end sequencing by ABlife, Inc (Wuhan, China).