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 and then sonicated using 8 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. 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 3 μg of amplified DNA from the antibody sample, no antibody, or IgG sample were vacuum desiccated and resuspended in 2.5 μL of H2O (Sigma W4502). 4.5 μL of 0.1 M NaHCO3 at pH 9 was added. The DNA was resuspended by vortexing several times. 2 μL of Cy3 or Cy5 dye (Amersham, PA23001 and PA25001 respectively) were added to the sample. The mix was incubated for 1.5 hours at room temperature in the dark. After incubation, 35 μL of 100 mM Na-Acetate pH5.2 was added and topped with H2O (Sigma W4502) to a total volume of 100 μL for each sample. The fluorescent labeled probes were purified using QIAquick PCR Purification kit (Qiagen 28106). The DNA was eluted with 50 μL of EB buffer from the kit, heated to 65°C. The 100 μL combined ChIP and no antibody samples were vacuum desiccated using a SpeedVac for 60 minutes at maximum heat.
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 and then sonicated using 8 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. 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 3 μg of amplified DNA from the antibody sample, no antibody, or IgG sample were vacuum desiccated and resuspended in 2.5 μL of H2O (Sigma W4502). 4.5 μL of 0.1 M NaHCO3 at pH 9 was added. The DNA was resuspended by vortexing several times. 2 μL of Cy3 or Cy5 dye (Amersham, PA23001 and PA25001 respectively) were added to the sample. The mix was incubated for 1.5 hours at room temperature in the dark. After incubation, 35 μL of 100 mM Na-Acetate pH5.2 was added and topped with H2O (Sigma W4502) to a total volume of 100 μL for each sample. The fluorescent labeled probes were purified using QIAquick PCR Purification kit (Qiagen 28106). The DNA was eluted with 50 μL of EB buffer from the kit, heated to 65°C. The 100 μL combined ChIP and no antibody samples were vacuum desiccated using a SpeedVac for 60 minutes at maximum heat.
Hybridization protocol
The mixed DNA was resuspended in 5 μL of H2O and mixed with 85 μL hybridization mix (100 μL of DIG Easy Hyb solution (Roche 603 558), 5 μL of 10 mg/mL calf thymus DNA (Sigma D8661), 5 μL of 10 mg/mL yeast tRNA (Invitrogen 15401-029)). The resulting solution was incubated at 65 °C for 2 minutes, then cooled to room temperature and applied to a CpG island array slide (UHN Microarray Centre HCGI12K) and incubated overnight at 42 °C in a hybridization chamber. After hybridization, the microarray slide was washed once with washing buffer 1 (1x SSC, 0.1% SDS) at 50°C for 5 minutes. This was followed by a wash with washing buffer 1 (room temperature for 5 minutes) and 2 washes with washing buffer 2 (0.2x SSC for 5 minutes) and one final wash with washing buffer 3 (0.1x SSC for 5 minutes at room temperature). The slide was then dried by spinning at 700 rpm for 15 minutes. To preserve the Cy5 dye from ozone-dependent degradation, which was observed during the summertime when the hybridization occurred, the slides were then dipped in DyeSaver 2 (Genisphere Q500600) and allowed to air dry.
Scan protocol
The microarray slides were scanned using a Gene Pix 4000B scanner (Axon Instruments) at multiple PMT voltages. Array images were first manually examined for image artifacts, and then quantitated using GenePix Pro (v6.0.1.27). A scan that maximized the dynamic range without saturating high-intensity signals was selected and carried forward for analysis. This scan was quantified using the “circular” segmentation algorithm.
All data were pre-processed using the variance-stabilizing normalization algorithm (VSN). VSN was implemented in the R statistical environment (v2.4.1) in the BioConductor open-source project. Version 1.12.0 of the vsn package was employed, with print-tip groups used as strata, and default parameterization except for using 1000 iterations for fitting (increased from the default of 10).
