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Sample GSM346087 Query DataSets for GSM346087
Status Public on Nov 24, 2009
Title MCF10A-MycChIPchip-ectopicMyc-rep7_chipA
Sample type genomic
 
Channel 1
Source name Myc ChIP-chip of exponentially growing MCF10A cells with ectopic MYC expression
Organism Homo sapiens
Characteristics CellLine: MCF10A, GrowthPhase: Exponential, EctopicGeneExpression: MYC, ChIPAntibody: Myc, ArrayBatch: 2
Treatment protocol Exponentially growing MCF10A cells were cross-linked with 1% formaldehyde for 15 min at 37°C. The cross-linking reaction was quenched by addition of glycine to a final concentration of 0.125 M for 15 min, followed by two washes with phosphate-buffered saline (PBS). Cells were resuspended in cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% [v/v] NP40, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) for 10 min on ice and then pelleted (5000 r.p.m., 5 min, 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 µg/ml aprotinin, 10 µg/ml leupeptin) for 10 min on ice and then sonicated with 11 pulses (setting high, 30 s per pulse, 30 s on ice between pulses) from a BioRuptor Sonicator (Diagenode, BioRuptor 200, UCD-200 TM-EX) to generate fragments between 100 and 500 bp. Lysates were centrifuged for 10 min at 1,000 rpm at 4°C. Supernatants were diluted into an equal volume with IP dilution buffer (0.01% SDS, 1.1% Triton-X100, 1.2 mN EDTA, 16.7 mM Tris–HCl pH 8.1, 0.2% Sarkosyl, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) and precleared for 30 min at 4°C with protein G-PLUS agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 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 MCF10A cells were incubated and rotated at 4°C for 12–16 h with each 1.5 µg normal rabbit IgG (Santa Cruz Biotechnology sc-2027), or home-made purified N262. 50 µl of salmon sperm DNA preblocked Protein G-PLUS agarose beads were added to each sample, incubated on a rotating platform at 4°C for 3 h. 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 min at room temperature then pelleted (3000 r.p.m., 30 s, 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 min, and then pelleted (3000 r.p.m., 3 min, 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 µl of TE (10 mM Tris, pH 7.5, 1 mM EDTA), 25 µl of 5x proteinase K buffer (1.25% SDS, 50 mM Tris, pH 7.5, 25 mM EDTA), and 1.5 µl of proteinase K (Roche 1413783) and incubated at 42°C for 2 h. Samples were then column purified sample using Qiagen Qiaquick PCR purification Kit, and eluted in 30 uL of Buffer EB.
Growth protocol MCF10A cells were grown asynchronously in a humidified incubator at 37 degrees C and 5% CO2 in a 1:1 mixture of Dulbecco's modified Eagle's medium and F12 medium (DMEM-F12) supplemented with 5% horse serum, hydrocortisone (0.5 ug/mL), insulin (10 ug/mL), epidermal growth factor (20 ng/mL), and cholera toxin (100 ng/mL).
Extracted molecule genomic DNA
Extraction protocol Entire ChIP samples were used for the library generation and subsequent amplification. For the library preparation and the amplification (round 1), GenomePlex Complete WGA kit was used (Sigma WGA2) as directed. Samples were then purified using QIAquick PCR Purification kit (Qiagen 28106) and 10 ng of then proceeded to the reamplification step (round 2). GenomePlex WGA amplification Kit was used (Sigma WGA3) as directed, and samples were then purified using QIAquick PCR Purification kit (Qiagen 28106).
Label Cy5
Label protocol WGA amplified DNA (4 µg) was hybridized to Agilent 2x244 promoter arrays at the UHN microarray center (Toronto, Canada). 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 min, then on ice for 5 min. 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 h. The enzyme was then inactivated at 65°C for 10 min. The labeled Genomic DNA was cleaned with Microcon columns (Millipore, Microcon YM-30) and eluted with 80.5 µl of 1x TE.
 
Channel 2
Source name IgG ChIP-chip of exponentially growing MCF10A cells with ectopic MYC expression
Organism Homo sapiens
Characteristics CellLine: MCF10A, GrowthPhase: Exponential, EctopicGeneExpression: MYC, ChIPAntibody: IgG, ArrayBatch: 2
Treatment protocol Exponentially growing MCF10A cells were cross-linked with 1% formaldehyde for 15 min at 37°C. The cross-linking reaction was quenched by addition of glycine to a final concentration of 0.125 M for 15 min, followed by two washes with phosphate-buffered saline (PBS). Cells were resuspended in cell lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% [v/v] NP40, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) for 10 min on ice and then pelleted (5000 r.p.m., 5 min, 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 µg/ml aprotinin, 10 µg/ml leupeptin) for 10 min on ice and then sonicated with 11 pulses (setting high, 30 s per pulse, 30 s on ice between pulses) from a BioRuptor Sonicator (Diagenode, BioRuptor 200, UCD-200 TM-EX) to generate fragments between 100 and 500 bp. Lysates were centrifuged for 10 min at 1,000 rpm at 4°C. Supernatants were diluted into an equal volume with IP dilution buffer (0.01% SDS, 1.1% Triton-X100, 1.2 mN EDTA, 16.7 mM Tris–HCl pH 8.1, 0.2% Sarkosyl, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin) and precleared for 30 min at 4°C with protein G-PLUS agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 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 MCF10A cells were incubated and rotated at 4°C for 12–16 h with each 1.5 µg normal rabbit IgG (Santa Cruz Biotechnology sc-2027), or home-made purified N262. 50 µl of salmon sperm DNA preblocked Protein G-PLUS agarose beads were added to each sample, incubated on a rotating platform at 4°C for 3 h. 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 min at room temperature then pelleted (3000 r.p.m., 30 s, 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 min, and then pelleted (3000 r.p.m., 3 min, 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 µl of TE (10 mM Tris, pH 7.5, 1 mM EDTA), 25 µl of 5x proteinase K buffer (1.25% SDS, 50 mM Tris, pH 7.5, 25 mM EDTA), and 1.5 µl of proteinase K (Roche 1413783) and incubated at 42°C for 2 h. Samples were then column purified sample using Qiagen Qiaquick PCR purification Kit, and eluted in 30 uL of Buffer EB.
Growth protocol MCF10A cells were grown asynchronously in a humidified incubator at 37 degrees C and 5% CO2 in a 1:1 mixture of Dulbecco's modified Eagle's medium and F12 medium (DMEM-F12) supplemented with 5% horse serum, hydrocortisone (0.5 ug/mL), insulin (10 ug/mL), epidermal growth factor (20 ng/mL), and cholera toxin (100 ng/mL).
Extracted molecule genomic DNA
Extraction protocol Entire ChIP samples were used for the library generation and subsequent amplification. For the library preparation and the amplification (round 1), GenomePlex Complete WGA kit was used (Sigma WGA2) as directed. Samples were then purified using QIAquick PCR Purification kit (Qiagen 28106) and 10 ng of then proceeded to the reamplification step (round 2). GenomePlex WGA amplification Kit was used (Sigma WGA3) as directed, and samples were then purified using QIAquick PCR Purification kit (Qiagen 28106).
Label Cy3
Label protocol WGA amplified DNA (4 µg) was hybridized to Agilent 2x244 promoter arrays at the UHN microarray center (Toronto, Canada). 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 min, then on ice for 5 min. 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 h. The enzyme was then inactivated at 65°C for 10 min. The labeled Genomic DNA was cleaned with Microcon columns (Millipore, Microcon YM-30) and eluted with 80.5 µl of 1x TE.
 
 
Hybridization protocol A 5 µg Cy5-labeled and 5 µg of Cy3-labeled DNAs were combined in a total volume of 158 µl. 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 min, and then incubated at 37°C for 30 min. 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 r.p.m. for 40 h. After hybridization the microarray slide was washed with Oligo aCGH/ChIP-on-chip Wash Buffer for 5 min at room temperature, followed by a wash for 5 min at 31°C.
Scan protocol Agilent Feature Extraction software v9.5 was used.
Description MCF10AMycRep07
Data processing Microarray data was scanned using the Agilent Feature Extraction Software (v9.5) and then loaded into the R statistical environment (v2.7.2) using the limma package (v2.14.7). Array data was pre-processed using variance-stabilizing normalization with default parameterization. Pre-processing employed the vsn package (v3.6.0) again in the R statistical environment. The raw and pre-processed data were subjected to a series of quality-control measures: all arrays were included in subsequent analyses. Significance-testing employed t-tests to compare the antibody and control channels, followed by an Empirical Baye’s moderation of standard error and a false-discovery rate adjustment for multiple-testing.
 
Submission date Nov 26, 2008
Last update date Nov 26, 2008
Contact name Paul C Boutros
E-mail(s) Paul.Boutros@utoronto.ca
Organization name Ontario Institute for Cancer Research
Street address 101 College Street, Suite 800
City Toronto
State/province Ontario
ZIP/Postal code M5G 0A3
Country Canada
 
Platform ID GPL4124
Series (2)
GSE13749 Genome-wide characterization of the transcriptional program of Myc-dependent transformation, ChIP-chip
GSE14264 Genome-wide characterization of the transcriptional program of Myc-dependent transformation

Data table header descriptions
ID_REF
VALUE VSN-normalized log ratios of Cy5/Cy3

Data table
ID_REF VALUE
1 -0.64
2 0.51
3 0.27
4 8.68490573474112e-03
5 0.17
6 8.60040217415925e-03
7 -0.31
8 0.60
9 0.43
10 0.09
11 -0.69
12 0.50
13 0.36
14 0.61
15 0.79
16 -0.50
17 0.25
18 0.19
19 0.52
20 -0.36

Total number of rows: 243494

Table truncated, full table size 2940 Kbytes.




Supplementary file Size Download File type/resource
GSM346087.tif.gz 116.1 Mb (ftp)(http) TIFF
GSM346087.txt.gz 68.5 Mb (ftp)(http) TXT
Processed data included within Sample table

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