Mouse embryonic stem Cells were cross-linked with 1% formaldehyde for 10 min at room temperature and formaldehyde was then inactivated by the addition of 125 mM glycine.
Extracted molecule
genomic DNA
Extraction protocol
Cells were cross-linked with 1% formaldehyde for 10 min at room temperature and formaldehyde was then inactivated by the addition of 125 mM glycine. Chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using anti-Klf2 (amino-acids 90-255 of mouse Klf2 raised in rabbit), anti-Klf4 (amino-acids 1-320 of mouse Klf4 raised in rabbit), anti-Klf5 (amino-acids 1-320 of mouse Klf5 raised in rabbit), anti-p53 (sc-100, Santa Cruz). Crosslinks were reversed overnight at 65C. ChIP DNA was purified by incubation with RNase A for 1 h at 37C, with 200 ug/ml Proteinase K for 2 h at 45C, phenol:chloroform:isoamyl alcohol extraction, and precipitation with 0.1 volumes of 3M sodium acetate, and 2 volumes of 100% ethanol.
Label
Cy5
Label protocol
Each of test and reference DNA samples (1 μg) was denatured in the presence of 5′ Cy3-or Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100U (exo-) Klenow fragment (New England Biolabs) and dNTP mixture [6 mM each in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 M EDTA, pH 8.0, precipitated with isopropanol, and resuspended in water. Then, 13 μg Cy5-labeled ChIP sample and 13 μg Cy3-labeled total sample were mixed, dried down, and resuspended in 40 μL hybridization buffer (NimbleGen Systems).
Mouse embryonic stem Cells were cross-linked with 1% formaldehyde for 10 min at room temperature and formaldehyde was then inactivated by the addition of 125 mM glycine.
Extracted molecule
genomic DNA
Extraction protocol
Cells were cross-linked with 1% formaldehyde for 10 min at room temperature and formaldehyde was then inactivated by the addition of 125 mM glycine. Chromatin extracts containing DNA fragments with an average size of 500 bp were immunoprecipitated using anti-Klf2 (amino-acids 90-255 of mouse Klf2 raised in rabbit), anti-Klf4 (amino-acids 1-320 of mouse Klf4 raised in rabbit), anti-Klf5 (amino-acids 1-320 of mouse Klf5 raised in rabbit), anti-p53 (sc-100, Santa Cruz). Crosslinks were reversed overnight at 65C. ChIP DNA was purified by incubation with RNase A for 1 h at 37C, with 200 ug/ml Proteinase K for 2 h at 45C, phenol:chloroform:isoamyl alcohol extraction, and precipitation with 0.1 volumes of 3M sodium acetate, and 2 volumes of 100% ethanol.
Label
Cy3
Label protocol
Each of test and reference DNA samples (1 μg) was denatured in the presence of 5′ Cy3-or Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100U (exo-) Klenow fragment (New England Biolabs) and dNTP mixture [6 mM each in TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 M EDTA, pH 8.0, precipitated with isopropanol, and resuspended in water. Then, 13 μg Cy5-labeled ChIP sample and 13 μg Cy3-labeled total sample were mixed, dried down, and resuspended in 40 μL hybridization buffer (NimbleGen Systems).
Hybridization protocol
After denaturation, hybridization was carried out in a MAUI® (MicroArray User Interface) Hybridization System ( BioMicro Systems, Salt Lake City, UT, USA) for 18 h at 42°C.
Scan protocol
The arrays were washed using wash buffer system (NimbleGen Systems), dried by centrifugation, and scanned at 5 μm resolution using the GenePix® 4000B scanner (Axon Instruments). Fluorescence intensity raw data were obtained from scanned images of arrays using NimbleScan™ 2.0 extraction software (NimbleGen Systems).
Description
107994 p53 ChIP-chip in E14 cells using p53 antibody
Data processing
The arrays were scanned at 5 μm resolution using the GenePix® 4000B scanner (Axon Instruments). Fluorescence intensity raw data were obtained from scanned images of arrays using NimbleScan™ 2.0 extraction software (NimbleGen Systems).
