GSM3102016: MCF10DCIS.COM.shWDR5.H3K4me3.Rep1; Homo sapiens; ChIP-Seq
ChIP lysates were generated from 10-15x10^6 cells. Briefly, after cross-linking (1% formaldehyde), cells were lysed in SDS buffer (100 mM NaCl, 50 mM Tris HCl pH 8.1, 5 mM EDTA pH 8, 0.5% SDS, and protease inhibitors) and sonicated in IP buffer (100 mM NaCl, 100 mM Tris-HCl pH 8.1, 5 mM EDTA pH 8, 0.3% SDS, 1.7% Triton X-100) prior to overnight incubation with antibodies against respective antibodies. DNA was eluted in TE containing 2% SDS and crosslinks reversed by incubation overnight at 65ºC. DNA was then purified by QIAquick columns (Qiagen) and quantified by using Qubit for dsDNA (Thermo Fisher Scientific). ChIP DNA was prepared for HiSeq 2000 Illumina sequencing using a standard protocol consisting in blunting, addition of dA overhangs, ligation of Illumina adapters, PCR with index primers and purification. A mixture of T4 DNA polymerase, DNA polymerase I and T4 kinase was used according to manufacturer's instruction. Library preparation is carried out on SPRIworks Fragment Library System.