HL60 (ATCC) cells were maintained in alpha-MEM with 10% FBS.
Extracted molecule
genomic DNA
Extraction protocol
Exponentially growing HL60 cells were cross-linked with 1% formaldehyde for 10 minutes at 37 °C. 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 phosphatebuffered 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 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 μg/mL aprotinin, 10 μg/mL leupeptin) for 10 minutes on ice. The sample was then sonicated using 11 pulses (setting high, 26 seconds per pulse, 26 seconds on ice between pulses) from a Model Diagenode BioRuptor Sonicator (Diagenode, BioRuptor 200, UCD-200 TM-EX) to generate fragments between 500 bp and 1000 bp. 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 μg/mL aprotinin, 10 μg/mL leupeptin) and precleared 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 salmonsperm 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: no-antibody control, 0.7 μg normal rabbit IgG (Santa Cruz Biotechnology sc-2027), 0.7 μg N262 (Santa Cruz Biotechnology sc-764), for the N262 home-made unpurified and purified antibodies, we determined empirically, by serial dilutions, the amount of antibody to be used. 50 μL 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). Crosslinks 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 hours. DNA was extracted from each sample using phenol:chloroform:isoamyl alcohol (25:24:1), then precipitated with 1/10th volume of 3 M sodium acetate (pH 5.3), 5 μg of glycogen, and 2 volumes of ethanol at -20 °C overnight. Pellets were collected by microcentrifugation and resuspended in 60 μL of H2O.
Label
Cy5
Label protocol
A total of 4 ug of WGA-amplified DNA was sent for labeling to the UHN microarray center (Toronto, Canada) and was labeld and hybridized to Agilent 2x244 promoter arrays according to Manufacturer's protocols.
Exponentially growing HL60 cells were cross-linked with 1% formaldehyde for 10 minutes at 37 °C. 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 phosphatebuffered 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 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 μg/mL aprotinin, 10 μg/mL leupeptin) for 10 minutes on ice. The sample was then sonicated using 11 pulses (setting high, 26 seconds per pulse, 26 seconds on ice between pulses) from a Model Diagenode BioRuptor Sonicator (Diagenode, BioRuptor 200, UCD-200 TM-EX) to generate fragments between 500 bp and 1000 bp. 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 μg/mL aprotinin, 10 μg/mL leupeptin) and precleared 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 salmonsperm 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: no-antibody control, 0.7 μg normal rabbit IgG (Santa Cruz Biotechnology sc-2027), 0.7 μg N262 (Santa Cruz Biotechnology sc-764), for the N262 home-made unpurified and purified antibodies, we determined empirically, by serial dilutions, the amount of antibody to be used. 50 μL 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). Crosslinks 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 hours. DNA was extracted from each sample using phenol:chloroform:isoamyl alcohol (25:24:1), then precipitated with 1/10th volume of 3 M sodium acetate (pH 5.3), 5 μg of glycogen, and 2 volumes of ethanol at -20 °C overnight. Pellets were collected by microcentrifugation and resuspended in 60 μL of H2O.
Label
Cy3
Label protocol
A total of 4 ug of WGA-amplified DNA was sent for labeling to the UHN microarray center (Toronto, Canada) and was labeld and hybridized to Agilent 2x244 promoter arrays according to Manufacturer's protocols.
Hybridization protocol
A total of 4 ug of WGA-amplified DNA was sent for labeling to the UHN microarray center (Toronto, Canada) and was labeld and hybridized to Agilent 2x244 promoter arrays according to Manufacturer's protocols.
Scan protocol
A total of 4 ug of WGA-amplified DNA was sent for labeling to the UHN microarray center (Toronto, Canada) and was labeld and hybridized to Agilent 2x244 promoter arrays according to Manufacturer's protocols. Images were quantified using Agilent Feature Extraction Software (version 9.5).
Description
Biological replicate 3 of 5. Growing HL60 cells chipped with Myc N262 antibody
Data processing
Microarray data was scanned using the Agilent Feature Extraction Software (v9.5) and then loaded into the R statistical environment (v2.6.2) using the limma package (v2.12.0). Array data was pre-processed using variance-stabilizing normalization, as above, with 1000 iterations to generate highly robust results on these large arrays. Pre-processing employed the vsn package (v3.2.1) 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.
