Cells were treated for 10 min with 1% p-formaldehyde and then with sodium glycine (100 mM) to inactivate p-formaldehyde. Cells were next washed twice with ice-cold PBS, detached and collected by centrifugation. The pellet was re-suspended in 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 3 mM CaCl2, 0.2% Triton X-100 and the Complete Proteinase Inhibitor Cocktail (Roche). Chromatin DNA was digested at 37ºC for 10 min using micrococcal nuclease (Roche) to generate approximately 80% mono-nucleosomal and 20% di-nucleosomal DNA fragments. The reactions were stopped using 5 mM EDTA. Digested chromatin was solubilized for 15 min on ice using 0.2% SDS. The pellet was removed by centrifugation (10,000xg, 20 min). For each chromatin sample a corresponding control fraction of the input DNA was prepared as follows. Genomic DNA samples (50 μg each) were incubated for 16 h at 65ºC with 0.5 NaCl. RNA and proteins were digested using 10 μg/ml RNAse A (37ºC; 15 min) and 1 mg/ml Proteinase K (50ºC; 1 h), respectively. DNA was purified using the DNA GenElute PCR Clean-up System. DNA fragmentation was confirmed using 2% agarose gel-electrophoresis. DNA concentration was measured at 260 nm.
Growth protocol
Human glioblastoma U251 and breast carcinoma MCF-7 cells (60-80% confluent) grown in DMEM supplemented with 10% fetal bovine serum (DMEM/FBS) were used in our experiments.
Extracted molecule
genomic DNA
Extraction protocol
Chromatin samples (100 μg each) were diluted in 500 μl ChIP buffer (50 mM HEPES, pH 7.5, containing 150 mM NaCl, 0.5% Triton X100, 1 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, and the Complete Proteinase Inhibitor Cocktail). The samples were pre-cleared by incubating for 1 h at 4ºC with 50 μl Protein G-coated magnetic Dynabeads suspension (Invitrogen). The H3ac, H3K4me2, H3K4me2 or H3K27me3 antibodies (10 μg each) and 50 μl Protein G-coated magnetic Dynabeads suspension were then added to the pre-cleared samples. Non-immune rabbit IgG was used as a control. To reduce non-specific binding, the beads were pre-treated for 1 h with the sonicated salmon sperm DNA (10 mg/ml). The final volume of the samples was made 600 μl using the ChIP buffer. ChIP samples were prepared in triplicate. The samples which did not contain the antibody were used as a control. The samples were then incubated at 4ºC for 12 h on a rotating platform. The beads were captured using a magnetic rack (Qiagen), washed 4 times in the ChIP buffer and twice in the ChIP buffer containing 0.5 M NaCl. The bound material was eluted from the beads at 37ºC for 30 min in 100 μl 0.1% NaHCO3-1% SDS. The soluble fraction was separated from the beads by centrifugation (10,000xg, 5 min). The soluble material was incubated 68ºC for 4 h in the presence of 0.5 M NaCl. RNA and proteins were digested using 10 μg/ml RNAse A (37ºC; 15 min) and 0.25 mg/ml Proteinase K (50ºC; 15 min), respectively. DNA was purified using the DNA GenElute PCR Clean-up System. DNA concentration was measured at 260 nM. The immunoprecipitated DNA samples (10 ng each) were amplified using the WGA2 whole genome amplification system. Amplification products were purified using the DNA GenElute PCR Clean-up System. DNA concentration was measured at 260 nM. The quality of the amplified DNA samples was confirmed using 2% agarose gel-electrophoresis.
Label
Cy5
Label protocol
The amplified IP DNA and WCE DNA samples (1 µg) were labeled with Cy5-dUTP and Cy3-dUTP, respectively, using Genomic DNA Enzymatic Labeling Kit (Agilent).
Cells were treated for 10 min with 1% p-formaldehyde and then with sodium glycine (100 mM) to inactivate p-formaldehyde. Cells were next washed twice with ice-cold PBS, detached and collected by centrifugation. The pellet was re-suspended in 20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 3 mM CaCl2, 0.2% Triton X-100 and the Complete Proteinase Inhibitor Cocktail (Roche). Chromatin DNA was digested at 37ºC for 10 min using micrococcal nuclease (Roche) to generate approximately 80% mono-nucleosomal and 20% di-nucleosomal DNA fragments. The reactions were stopped using 5 mM EDTA. Digested chromatin was solubilized for 15 min on ice using 0.2% SDS. The pellet was removed by centrifugation (10,000xg, 20 min). For each chromatin sample a corresponding control fraction of the input DNA was prepared as follows. Genomic DNA samples (50 μg each) were incubated for 16 h at 65ºC with 0.5 NaCl. RNA and proteins were digested using 10 μg/ml RNAse A (37ºC; 15 min) and 1 mg/ml Proteinase K (50ºC; 1 h), respectively. DNA was purified using the DNA GenElute PCR Clean-up System. DNA fragmentation was confirmed using 2% agarose gel-electrophoresis. DNA concentration was measured at 260 nm.
Growth protocol
Human glioblastoma U251 and breast carcinoma MCF-7 cells (60-80% confluent) grown in DMEM supplemented with 10% fetal bovine serum (DMEM/FBS) were used in our experiments.
Extracted molecule
genomic DNA
Extraction protocol
Chromatin samples (100 μg each) were diluted in 500 μl ChIP buffer (50 mM HEPES, pH 7.5, containing 150 mM NaCl, 0.5% Triton X100, 1 mM EDTA, 0.1% sodium deoxycholate, 0.1% SDS, and the Complete Proteinase Inhibitor Cocktail). The samples were pre-cleared by incubating for 1 h at 4ºC with 50 μl Protein G-coated magnetic Dynabeads suspension (Invitrogen). The H3ac, H3K4me2, H3K4me2 or H3K27me3 antibodies (10 μg each) and 50 μl Protein G-coated magnetic Dynabeads suspension were then added to the pre-cleared samples. Non-immune rabbit IgG was used as a control. To reduce non-specific binding, the beads were pre-treated for 1 h with the sonicated salmon sperm DNA (10 mg/ml). The final volume of the samples was made 600 μl using the ChIP buffer. ChIP samples were prepared in triplicate. The samples which did not contain the antibody were used as a control. The samples were then incubated at 4ºC for 12 h on a rotating platform. The beads were captured using a magnetic rack (Qiagen), washed 4 times in the ChIP buffer and twice in the ChIP buffer containing 0.5 M NaCl. The bound material was eluted from the beads at 37ºC for 30 min in 100 μl 0.1% NaHCO3-1% SDS. The soluble fraction was separated from the beads by centrifugation (10,000xg, 5 min). The soluble material was incubated 68ºC for 4 h in the presence of 0.5 M NaCl. RNA and proteins were digested using 10 μg/ml RNAse A (37ºC; 15 min) and 0.25 mg/ml Proteinase K (50ºC; 15 min), respectively. DNA was purified using the DNA GenElute PCR Clean-up System. DNA concentration was measured at 260 nM. The immunoprecipitated DNA samples (10 ng each) were amplified using the WGA2 whole genome amplification system. Amplification products were purified using the DNA GenElute PCR Clean-up System. DNA concentration was measured at 260 nM. The quality of the amplified DNA samples was confirmed using 2% agarose gel-electrophoresis.
Label
Cy3
Label protocol
The amplified IP DNA and WCE DNA samples (1 µg) were labeled with Cy5-dUTP and Cy3-dUTP, respectively, using Genomic DNA Enzymatic Labeling Kit (Agilent).
Hybridization protocol
Labeled DNAs were hybridized with custom microarrays for 40 hrs at 65°C.
Scan protocol
Scanned on an Agilent G2565AA scanner.
Description
none
Data processing
Intensity data was extracted using Feature Extraction software v.10.5 (Agilent) and normalized using linear method. Data table was assembled in DNA Analytics 4.0.76 software (Agilent).
