Instrument: Illumina Genome Analyzer IIx
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody. The Chromatin immunoprecipitated DNA fragments (approx.10 ng in 30 µl water) are end repaired by using T4 DNA polymerase, Klenow DNA polymerase and T4 polynucleotide kinase in the presence of dNTPs. And an ‘A’ base is added to the 3’end of the blunt phosphorylated DNA fragments, using the polymerase activity of Klenow fragment. Then, adaptors (Adaptor oligo mix, Illumina) are ligated to the ends of the DNA fragments. The resulting DNA fragments are purified by MinElute column (QIAGEN). 300 ~ 500 bp DNA fragments are purified by using E-Gel SizeSelect agarose Gels (Invitrogen) according to manufacturer’s instruction. Size selected adaptor-modified DNA fragments are amplified by 18 cycles of PCR using Phusion polymerase and PCR primer 1.1 and 2.1 (Illumina). The resulting PCR products are purified by MinElute column (QIAGEN). The size and amount of DNA fragments (libraries) are validated by Bioanalyzer (Agilent).