Seven-week old female severe combined immunodefiicent (SCID) mice (Ontario Cancer Institute) were sub-cutaneously injected into the right flank with 5 x 106 A549, MGH8, MGH24 or RVH6849 cells suspended in 0.1 mL of sterile PBS. Mice were monitored daily. At maximal tumor burden, mice were sacrificed by CO2 inhalation and tumors were excised and immediately snap-frozen in liquid N2 for later analysis.
Extracted molecule
genomic DNA
Extraction protocol
0.03g of snap-frozen tissue per antibody was chopped into small pieces in 1X PBS, formaldehyde was added to a final concentration of 1% at room temperature for 15 minutes. The crosslinking reaction was quenched by addition of glycine to a final concentration of 0.125 M for 5 minutes, followed by two washes with phosphate-buffered saline (PBS). Samples were washed one time in 1X PBS. The tissues were disaggregated in 1X PBS using a dounce homogenizer, Brinkmann Homogenizer. Model PT 10/35 (110volts, 6Amps, 60Hz). Polytron. Switzerland. Cells were resuspended in cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% [v/v] NP40, 1 mM PMSF, 10 g/mL aprotinin, 10 g/mL leupeptin) for 10 minutes on ice and then pelleted (5000 rpm, 5 minutes, 4 C). The pellet was resuspended in 1 mL of nuclei lysis buffer (50mM Tris-HCl pH 8.1, 10mM EDTA, 1% SDS, 1 mM PMSF, 10 g/mL aprotinin, 10 g/mL leupeptin) for 10 minutes on ice. Prior to sonication, 0,1g of glass beads (Sigma G-1277) were added to each sample. The samples were sonicated on ice at setting 7 pulses (12-13 Watts, setting 10, 10 seconds per pulse, 45 seconds on ice between pulses) from a Model 60 Sonic Dismembrator (Fisher Scientific 15-338-53) to generate fragments between 600 bp and 1000 bp and then were microcentrifuged. Lysates were centrifuged for 10 minutes at 21,000 x g at 4 °C. Supernatants were diluted into an equal volume with IP dilution buffer (0.01% SDS, 1.1% Triton-X100, 1.2mN EDTA, 16.7mM Tris-HCl pH 8.1, 0.2% Sarkosyl, 1 mM PMSF, 10 g/mL aprotinin, 10 g/mL leupeptin) and pre-cleared for 30 minutes at 4 °C with protein G-PLUS agarose beads (Santa Cruz Biotechnology sc-2002). Prior to use, G-PLUS agarose beads were blocked with salmon sperm DNA at a final concentration of 50 µg/mL and rotated overnight at 4 °C. Diluted and cleared extracts corresponding to 10 x 106 HL60 cells were incubated and rotated at 4°C for approximately 12 to 16 hours with each of the following antibodies: 0.7 µg normal rabbit IgG (Santa Cruz Biotechnology sc-2027), 0.7 µg N262 home made, as described elsewhere (Ponzielli et al. NAR.2008. 36(21):e144). 50 L of salmon sperm DNA pre-blocked Protein G-PLUS agarose beads were added to each sample, incubated on a rotating platform at 4 °C for 3 hours. Each pellet was washed once with 1.4 mL of sonication buffer and then twice with 1.4 mL of high salt buffer (0.1% [v/v] SDS, 1% [v/v] Triton X-100, 1 mM EDTA, 50 mM HEPES, 500 mM NaCl, 0.1% [w/v] sodium deoxycholate) and then once with 1.4 mL LiCl Buffer (250 mM LiCl, 1% [v/v] NP-40, 1% [w/v] sodium deoxycholate, 1 mM EDTA, 1 mM Tris pH 8) and finally twice with 1.4 mL TE pH 8 (10 mM Tris pH8, 1 mM EDTA). For each wash, the pellets were mixed for 5 minutes at room temperature then pelleted (3000 rpm, 30 seconds, room temperature). After the last wash, the pellets were eluted in 300 µL of Elution buffer (1% [w/v] SDS, 10 mM Tris pH 8, 5 mM EDTA), incubated at 65 °C for 15 minutes, and then pelleted (3000 rpm, 3 minutes, room temperature). Cross-links were reversed in the presence of 200 mM NaCl at 65 °C over-night and samples were treated with RNase A (Sigma R5500). After ethanol precipitation, the samples were resuspended in 100 µ of TE (10 mM Tris, pH 7.5, 1 mM EDTA), 25 µ of 5x proteinase K buffer (1.25% SDS, 50 mM Tris, pH 7.5, 25 mM EDTA), and 1.5 µ of proteinase K (Roche 1413783) and incubated at 42 °C for 2 hours. DNA was isolated using the QIAquick PCR purification kit (Qiagen 28106) and resuspended in 60 µ of H2O. The DNA amplification was performed following a protocol described elsewhere (O'Geen et al. Biotechniques 2006. 41: 577-580) This method efficiently primes the DNA fragments to generate a library of DNA fragments with defined 3' and 5' termini. The library is then replicated using linear amplification in the initial steps followed by a limited round of geometric amplifications. Entire ChIP samples with an average size of 500-1,000 bp of DNA fragments were used for the library generation and subsequent amplification. For the library preparation and the amplification (round 1), GenomePlex Complete Whole Genome Amplification (WGA) kit was used (Sigma WGA2) as follows. Library Preparation step: 2 L of Library Preparation Buffer was added to the ChIP material that has been concentrated to 10 L. 1 L of Library Stabilization Solution was added and heated at 95 °C for 2 minutes in a thermal cycler and immediately cooled on ice. 1 L of Library Preparation Enzyme was added and incubated in a thermal cycler precooled to 16 °C (16 °C for 20 minutes, 24 °C for 20 minutes, 37 °C for 20 minutes, 75 °C for 5 minutes). Amplification step (round 1): the following master mix was added to each sample prepared on the previous step, 7.5 L of 10x Amplification Master Mix, 47.5 L of nuclease-free H2O and 5 L of WGA DNA Polymerase. Samples were incubated in a thermal cycler (95 °C for 3 minutes, then 14 cycles of 94 °C for 15 seconds, and 65 °C for 5 minutes). Samples were then purified using QIAquick PCR Purification kit (Qiagen 28106). 10 ng of each amplified sample was tested for specificity as described above under the PCR title. For the reamplification step (round 2), GenomePlex WGA amplification Kit was used (Sigma WGA3). A master mix of 7.5 L of 10x Amplification Master Mix, 47.5 L of nuclease-free H2O and 5 L of WGA DNA Polymerase was added to 15 ng of purified amplification product previously diluted in nuclease-free H2O to 10 L volume. Samples were then purified using QIAquick PCR Purification kit (Qiagen 28106). Amplified sample (10 ng) was tested for specificity as described above. WGA amplified DNA (4 g) was hybridized to Agilent 2x244 promoter arrays at the UHN microarray center (Toronto, Canada). Based on our previous results a single batch of arrays was used with lot number 19585.
Label
Cy5
Label protocol
Arrays were labeled using the Agilent Genomic DNA Labelling Kit and hybridized using the aCGH Hybridization Kit following the ChIP-on-chip v10 protocol as follows: for each 1x244 promoter array, 2 µg of DNA was brought to 26 µL final volume. 5 µL of Random Primers (supplied with Agilent Genomic DNA Labeling Kit PLUS) was added and the mix was incubated at 95°C for 3 minutes, then on ice for 5 minutes. The 31 µL were mixed with the Labeling Mix to a final volume of 50 µL (10 µL of 5x Buffer, 5 µL of 10x dNTP, 3 µL of 1 mM Cyanine 3-UTP or 1 mM Cyanine 5-dUTP, 1 µL of Exo-Klenow fragment) and incubated at 37 °C for 2 hours. The enzyme was then inactivated at 65°C for 10 minutes. The labeled Genomic DNA was cleaned with Microcon columns (Millipore, Microcon YM-30) and eluted with 80.5 µL of 1x TE. 5 µg Cy5-labeled and 5 µg of Cy3-labeled DNAs were combined in a total volume of 158 µL.
Channel 2
Source name
DNA ChIPed from MGH8 Xenografts using a non-specific IgG antibody
Seven-week old female severe combined immunodefiicent (SCID) mice (Ontario Cancer Institute) were sub-cutaneously injected into the right flank with 5 x 106 A549, MGH8, MGH24 or RVH6849 cells suspended in 0.1 mL of sterile PBS. Mice were monitored daily. At maximal tumor burden, mice were sacrificed by CO2 inhalation and tumors were excised and immediately snap-frozen in liquid N2 for later analysis.
Extracted molecule
genomic DNA
Extraction protocol
0.03g of snap-frozen tissue per antibody was chopped into small pieces in 1X PBS, formaldehyde was added to a final concentration of 1% at room temperature for 15 minutes. The crosslinking reaction was quenched by addition of glycine to a final concentration of 0.125 M for 5 minutes, followed by two washes with phosphate-buffered saline (PBS). Samples were washed one time in 1X PBS. The tissues were disaggregated in 1X PBS using a dounce homogenizer, Brinkmann Homogenizer. Model PT 10/35 (110volts, 6Amps, 60Hz). Polytron. Switzerland. Cells were resuspended in cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% [v/v] NP40, 1 mM PMSF, 10 g/mL aprotinin, 10 g/mL leupeptin) for 10 minutes on ice and then pelleted (5000 rpm, 5 minutes, 4 C). The pellet was resuspended in 1 mL of nuclei lysis buffer (50mM Tris-HCl pH 8.1, 10mM EDTA, 1% SDS, 1 mM PMSF, 10 g/mL aprotinin, 10 g/mL leupeptin) for 10 minutes on ice. Prior to sonication, 0,1g of glass beads (Sigma G-1277) were added to each sample. The samples were sonicated on ice at setting 7 pulses (12-13 Watts, setting 10, 10 seconds per pulse, 45 seconds on ice between pulses) from a Model 60 Sonic Dismembrator (Fisher Scientific 15-338-53) to generate fragments between 600 bp and 1000 bp and then were microcentrifuged. Lysates were centrifuged for 10 minutes at 21,000 x g at 4 °C. Supernatants were diluted into an equal volume with IP dilution buffer (0.01% SDS, 1.1% Triton-X100, 1.2mN EDTA, 16.7mM Tris-HCl pH 8.1, 0.2% Sarkosyl, 1 mM PMSF, 10 g/mL aprotinin, 10 g/mL leupeptin) and pre-cleared for 30 minutes at 4 °C with protein G-PLUS agarose beads (Santa Cruz Biotechnology sc-2002). Prior to use, G-PLUS agarose beads were blocked with salmon sperm DNA at a final concentration of 50 µg/mL and rotated overnight at 4 °C. Diluted and cleared extracts corresponding to 10 x 106 HL60 cells were incubated and rotated at 4°C for approximately 12 to 16 hours with each of the following antibodies: 0.7 µg normal rabbit IgG (Santa Cruz Biotechnology sc-2027), 0.7 µg N262 home made, as described elsewhere (Ponzielli et al. NAR.2008. 36(21):e144). 50 L of salmon sperm DNA pre-blocked Protein G-PLUS agarose beads were added to each sample, incubated on a rotating platform at 4 °C for 3 hours. Each pellet was washed once with 1.4 mL of sonication buffer and then twice with 1.4 mL of high salt buffer (0.1% [v/v] SDS, 1% [v/v] Triton X-100, 1 mM EDTA, 50 mM HEPES, 500 mM NaCl, 0.1% [w/v] sodium deoxycholate) and then once with 1.4 mL LiCl Buffer (250 mM LiCl, 1% [v/v] NP-40, 1% [w/v] sodium deoxycholate, 1 mM EDTA, 1 mM Tris pH 8) and finally twice with 1.4 mL TE pH 8 (10 mM Tris pH8, 1 mM EDTA). For each wash, the pellets were mixed for 5 minutes at room temperature then pelleted (3000 rpm, 30 seconds, room temperature). After the last wash, the pellets were eluted in 300 µL of Elution buffer (1% [w/v] SDS, 10 mM Tris pH 8, 5 mM EDTA), incubated at 65 °C for 15 minutes, and then pelleted (3000 rpm, 3 minutes, room temperature). Cross-links were reversed in the presence of 200 mM NaCl at 65 °C over-night and samples were treated with RNase A (Sigma R5500). After ethanol precipitation, the samples were resuspended in 100 µ of TE (10 mM Tris, pH 7.5, 1 mM EDTA), 25 µ of 5x proteinase K buffer (1.25% SDS, 50 mM Tris, pH 7.5, 25 mM EDTA), and 1.5 µ of proteinase K (Roche 1413783) and incubated at 42 °C for 2 hours. DNA was isolated using the QIAquick PCR purification kit (Qiagen 28106) and resuspended in 60 µ of H2O. The DNA amplification was performed following a protocol described elsewhere (O'Geen et al. Biotechniques 2006. 41: 577-580) This method efficiently primes the DNA fragments to generate a library of DNA fragments with defined 3' and 5' termini. The library is then replicated using linear amplification in the initial steps followed by a limited round of geometric amplifications. Entire ChIP samples with an average size of 500-1,000 bp of DNA fragments were used for the library generation and subsequent amplification. For the library preparation and the amplification (round 1), GenomePlex Complete Whole Genome Amplification (WGA) kit was used (Sigma WGA2) as follows. Library Preparation step: 2 L of Library Preparation Buffer was added to the ChIP material that has been concentrated to 10 L. 1 L of Library Stabilization Solution was added and heated at 95 °C for 2 minutes in a thermal cycler and immediately cooled on ice. 1 L of Library Preparation Enzyme was added and incubated in a thermal cycler precooled to 16 °C (16 °C for 20 minutes, 24 °C for 20 minutes, 37 °C for 20 minutes, 75 °C for 5 minutes). Amplification step (round 1): the following master mix was added to each sample prepared on the previous step, 7.5 L of 10x Amplification Master Mix, 47.5 L of nuclease-free H2O and 5 L of WGA DNA Polymerase. Samples were incubated in a thermal cycler (95 °C for 3 minutes, then 14 cycles of 94 °C for 15 seconds, and 65 °C for 5 minutes). Samples were then purified using QIAquick PCR Purification kit (Qiagen 28106). 10 ng of each amplified sample was tested for specificity as described above under the PCR title. For the reamplification step (round 2), GenomePlex WGA amplification Kit was used (Sigma WGA3). A master mix of 7.5 L of 10x Amplification Master Mix, 47.5 L of nuclease-free H2O and 5 L of WGA DNA Polymerase was added to 15 ng of purified amplification product previously diluted in nuclease-free H2O to 10 L volume. Samples were then purified using QIAquick PCR Purification kit (Qiagen 28106). Amplified sample (10 ng) was tested for specificity as described above. WGA amplified DNA (4 g) was hybridized to Agilent 2x244 promoter arrays at the UHN microarray center (Toronto, Canada). Based on our previous results a single batch of arrays was used with lot number 19585.
Label
Cy3
Label protocol
Arrays were labeled using the Agilent Genomic DNA Labelling Kit and hybridized using the aCGH Hybridization Kit following the ChIP-on-chip v10 protocol as follows: for each 1x244 promoter array, 2 µg of DNA was brought to 26 µL final volume. 5 µL of Random Primers (supplied with Agilent Genomic DNA Labeling Kit PLUS) was added and the mix was incubated at 95°C for 3 minutes, then on ice for 5 minutes. The 31 µL were mixed with the Labeling Mix to a final volume of 50 µL (10 µL of 5x Buffer, 5 µL of 10x dNTP, 3 µL of 1 mM Cyanine 3-UTP or 1 mM Cyanine 5-dUTP, 1 µL of Exo-Klenow fragment) and incubated at 37 °C for 2 hours. The enzyme was then inactivated at 65°C for 10 minutes. The labeled Genomic DNA was cleaned with Microcon columns (Millipore, Microcon YM-30) and eluted with 80.5 µL of 1x TE. 5 µg Cy5-labeled and 5 µg of Cy3-labeled DNAs were combined in a total volume of 158 µL.
Hybridization protocol
The 158 µL were mixed with 50 µL of 1 mg/mL of Human Cot-1 DNA, 52 µL of 10x Agilent Blocking Agent, 260 µL of 2x Agilent Hybridization Buffer. Samples were heated at 95°C for 3 minutes, and then incubated at 37°C for 30 minutes. 490 µL of the sample were applied to the 1x244 promoter array assembled in a chamber and incubated in a rotisserie hybridization oven at 65°C and 20 RPM for 40 hours. After hybridization the microarray slide was washed with Oligo aCGH/ChIP-on-chip Wash Buffer for 5 minutes at room temperature, followed by a wash for 5 minutes at 31°C. QC metrics used were based on the ChIP-on-chip requirements and no arrays were removed or repeated.
Scan protocol
Microarray data was scanned using a G2565BA DNA Scanner; Agilent Scan Control software (v7.0) with the following settings: 5 um scan, 100% PMT (both channels), 100% laser power (both channels), scan area 61 x 21.6 mm.
Description
NA
Data processing
Raw microarray data were loaded into the limma package (v2.12.0) of the BioConductor open-source library for the R statistical environment (v2.6.2). Data were assessed for spatial and distributional homogeneity; no arrays were excluded. Pre-processing used the variance-stabilizing normalization (vsn) algorithm, as implemented in the vsn package (v3.2.1) of BioConductor using 10,000 iterations to ensure convergence.