About 1 x 10e7 cells were cross-linked with 1 % formaldehyde, lysed, and sonicated with a Bioruptor UCD-250 (Cosmo Bio, Tokyo, Japan).
Label
Cy3
Label protocol
Cross-link was reversed at 65℃, DNA was recovered with RNaseA and proteinase K treatment, followed by phenol extraction and ethanol precipitation, and dissolved in 100 μl of 1 x TE. 500 ng of DNA was labeled using Agilent Genomic DNA labeling Kit PLUS (5188-5309).
cell line: Normal prostatic epithelial cell line, RWPE1 antibody: H3K9me3 cell fraction: IP
Extracted molecule
genomic DNA
Extraction protocol
About 1 x 10e7 cells were cross-linked with 1 % formaldehyde, lysed, and sonicated with a Bioruptor UCD-250 (Cosmo Bio, Tokyo, Japan).
Label
Cy5
Label protocol
Solubilized chromatin was diluted 10-fold in dilution buffer [50 mM Tris-HCl, pH 8.0, 167 mM NaCl, 1.1 % (w/v) Triton X-100, 0.11 % (w/v) sodium deoxycholate (DOC)], and incubated with antibody against H3K9me3 (07-442, Millipore) at 4ºC overnight. Immuno-complexes were collected with Dynabeads Protein A, washed with 1 x RIPA buffer [50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 % (w/v) Triton X-100, 0.1 % (w/v) SDS, 0.1 % (w/v) DOC] containing 150 mM NaCl, 1 x RIPA buffer containing 500 mM NaCl, LiCl wash buffer [10 mM Tris-HCl, pH 8.0, 0.25 M LiCl, 1 mM EDTA, 0.5 % (w/v) NP-40, 0.5 % (w/v) DOC] and 1 x TE containing 50 mM NaCl. After cross-link reversal, DNA was recovered with RNaseA and proteinase K treatment, followed by phenol extraction and ethanol precipitation, and dissolved in 100 μl of 1 x TE. 500 ng of DNA was labeled using Agilent Genomic DNA labeling Kit PLUS (5188-5309).
Hybridization protocol
Labelled DNA mixed with cot-1 DNA and Agilent blocking solution was hybridized to microarray for 40 hours at 67℃ and 10 rpm rotation. Microarrays was washed according to Agilent protocol.
Scan protocol
The microarray was scanned with an Agilent G2565BA microarray scanner (Agilent Technologies).
Description
To recover immunocomplex, Dynabeads protein A (Dynal) was used.
Data processing
Scanned data was analyzed with Feature Extraction Ver.9.1 and normalized (Background subtraction) using Agilent G4477AA ChIP Analytics 1.3 software.
