1% formaldehyde was added to ~1 × 10^8 cells for 10 min at 37°C. Cells were washed twice in cold phosphate-buffered saline with protease inhibitors and then placed in hypotonic buffer for 20 min, followed by Dounce homogenization to isolate cross-linked nuclei. Nuclei were placed in sodium dodecyl sulfate lysis buffer for 30 min and then sonicated on ice with 21 10-s pulses, each followed by a 10-s rest period. Samples were diluted and then precleared at 4°C for 60 min with protein A- or G- agarose beads. Samples were immunoprecipitated for 12 to 18 h on a rotating platform at 4°C. Antibodies utilized for immunoprecipitation included histone 3 monomethyl lysine 27 (H3K27me1, Upstate 07-448), histone 3 tri-methyl lysine 4 (H3K4me3, ABCAM, ab8580), and nonspecific rabbit IgG (Santa Cruz, sc-2091). Antibody-bound DNA-protein complexes were collected using protein A- or G-agarose beads, washed, eluted from the beads, and cross-linking of DNA-protein adducts reversed by incubation at 65 °C for 4 h. DNA was cleaned with the QIAquick PCR purification kit (Qiagen) according to manufacturer’s instructions and amplified with the GenomePlex Whole Genome Amplification kit (Sigma) according to manufacturer’s instructions. Amplified DNA was cleaned using the QIAquick PCR purification kit (Qiagen) before amplification, labeling, and hybridization to arrays.
Label
cy5
Label protocol
Labelling was performed by NimbleGen using standard Nimblegen protocols
1% formaldehyde was added to ~1 × 10^8 cells for 10 min at 37°C. Cells were washed twice in cold phosphate-buffered saline with protease inhibitors and then placed in hypotonic buffer for 20 min, followed by Dounce homogenization to isolate cross-linked nuclei. Nuclei were placed in sodium dodecyl sulfate lysis buffer for 30 min and then sonicated on ice with 21 10-s pulses, each followed by a 10-s rest period. Samples were diluted and then precleared at 4°C for 60 min with protein A- or G- agarose beads. Samples were immunoprecipitated for 12 to 18 h on a rotating platform at 4°C. Antibodies utilized for immunoprecipitation included histone 3 monomethyl lysine 27 (H3K27me1, Upstate 07-448), histone 3 tri-methyl lysine 4 (H3K4me3, ABCAM, ab8580), and nonspecific rabbit IgG (Santa Cruz, sc-2091). Antibody-bound DNA-protein complexes were collected using protein A- or G-agarose beads, washed, eluted from the beads, and cross-linking of DNA-protein adducts reversed by incubation at 65 °C for 4 h. DNA was cleaned with the QIAquick PCR purification kit (Qiagen) according to manufacturer’s instructions and amplified with the GenomePlex Whole Genome Amplification kit (Sigma) according to manufacturer’s instructions. Amplified DNA was cleaned using the QIAquick PCR purification kit (Qiagen) before amplification, labeling, and hybridization to arrays.
Label
cy3
Label protocol
Labelling was performed by NimbleGen using standard Nimblegen protocols
Hybridization protocol
Hybridization was performed by NimbleGen using standard Nimblegen protocols
Scan protocol
Scanning was performed by NimbleGen using standard Nimblegen protocols
Data processing
Log2 transformed Control and immunoprecipitation data files were processed with the R Smudgekit version 2.4 software to remove potential chip hybridization artifacts. The average values of the two replicate oligonucleotide probes on each array was used for all processing. All available control data was quantile normalized together, and the median value for each probe was used as the input value. The log2 difference (ChIP/control) is reported.
