GSM1647620: 155N; Homo sapiens; ChIP-Seq
Cell or Chopped tissue pellets were homogenized in PBS containing proteinase inhibitor. The nuclei were checked by phase-contrast microscopy to maximize the yield of nuclei (~5x107). With sufficient lysis in 25% sucrose, 0.2% NP-40/TBS, the pellets were layered on 50% [w/v] sucrose/TBS, and then centrifuged at 21,000 g, 30 min, 4°C. The nuclei pellets were resuspended in digestion buffer (0.32 M sucrose, 50 mM Tris-HCl (pH 7.5), 4 mM MgCl2, 1 mM CaCl2), and digested with MNase for ~10 min to generate mainly mononucleosomes that were immunoprecipitated with anti-H3K4me3 antibody (ab8580, Abcam). After incubation over night, Protein A Dynal magnetic beads were washed 6 times using modified RIPA buffer (50 mM Tris-HCL pH 7.8, 1 mM EDTA, 0.25% Na Deoxycholate, 1% NP-40, 0.5 M LiCl). The DNA was eluted in 1% SDS and 0.1M NaHCO3 and reversed cross-linking at 65°C, over night. The library was generated using NEBNext ChIP-Seq Library Prep Master Mix Set, and amplified with 10 PCR cycles. The library was prepared by size selection for mononucleosome fragments.