GSM6593325: HepG2_UHRF1-TTD_CIDOP_bio-rep2; Homo sapiens; ChIP-Seq
The UHRF1-TTD CIDOP-seq and α-H3K9me2 ChIP-seq experiments were performed in biological duplicates. Mononucleosome generation and histone precipitation were performed with a modular protocol, all parameters optimized for each enrichment reagent. Briefly, the cells were resuspended in lysis buffer and digested with MNase in one tube, centrifuged, and the supernatant containing mononucleosomes collected, flash-frozen and stored in -80 °C. UHRF1-TTD or α-H3K9me2 (ab1220) were first incubated with appropriate magnetic beads (GST-Pierce magnetic or DynabeadsG 10004D) for 2 h, and then with precleared chromatin for overnight binding. Beads were washed three times with PB200 (Tris-HCl pH 8.0 50 mM, NaCl 200 mM,  DTT 2mM, Igepal CA-360 0.5%), followed by two rinse steps (Tris-HCl pH 8.0 10 mM or Tris-HCl pH 8.0 10 mM, LiCl 150 mM). Then they were eluted (Tris-HCl pH 8.0 50 mM, NaCl 50 mM, EDTA 5mM, SDS 1%), digested with Proteinase K, and purified with ChIP DNA Purification Kit (Active Motif). NGS libraries were prepared with 10 ng DNA using the NEBNext® Ultra™ II DNA Library Prep Kit according to the manufacturer's instructions