GSM1624501: 309M3-B lot1_ChIPSeq experiment 1; Homo sapiens; ChIP-Seq
Native ChIP experiments using HEK293 cells were performed as previously described (Brand M. et al., Nat. Protoc., 3, 398-409 (2008) and Ruthenburg, A.J. et al., Cell, 145, 692-706 (2011)) except for the following modifications. Micrococcal nuclease-digested nuclei from HEK293 cells were purified using hydroxyapatite chromatography with stepwise elution in 50, 100, 200 and 500 mM sodium phosphate buffer (pH 7.2) containing 100 mM NaCl, 1 mM EDTA and 200 uM PMSF. The purity of nucleosomes in elution fractions was examined by SDS-PAGE and high-purity fractions were used for ChIP. The antibody-coated beads were prepared by incubating 132 uL of Dynabeads M280 streptavidin (Life Technology) and 3.3 ug of a biotinylated Fab antibody (molar equivalent to 1 ug of IgG antibody) for 1hr at 4 ˚C. Excess biotin-binding sites of streptavidin were blocked with biotin. The antibody-coated beads were incubated with 10 ug of purified nucleosomes at 4˚C for overnight. Washing, elution and DNA purification were performed as previously described (Brand M. et al., Nat. Protoc., 3, 398-409 (2008)). Library preparation for sequencing was performed using TruSeq ChIP sample preparation Kit, Set A (Illumina) according to the manufacturer’s protocol.