Drosophila Kc cells were grown in BPYE medium (Shields and Sang M3 Insect medium, Sigma, supplemented with 25% w/v bactopeptone, 20% w/v yeast extract, 5% heat inactivated fetal calf serum, all Gibco) at 23degC
Extracted molecule
genomic DNA
Extraction protocol
The ChIP method was and adaptation of a previously published protocol (Rietveld, L.E., Caldenhoven, E. & Stunnenberg, H.G. In vivo repression of an erythroid-specific gene by distinct corepressor complexes. Embo J 21, 1389-97, 2002). Approximately 2x108 Kc cells were cross-linked by the addition of formaldehyde (1% final concentration). The reaction was quenched with 125 mM Glycine and the cells were washed twice with PBS. Subsequently, the cells were incubated 5� on ice in 1.5 ml lysis buffer (1% v/v SDS, 10 mM EDTA pH8.0, 50 mM Tris-HCl pH8.0, protease inhibitor cocktail - Roche). The cell lysates were sonicated and centrifuged to remove cell debris. We used the equivalent of 1 x 10E6 cells for one immunoprecipitation. Chromatin samples were diluted 10-fold in IP buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8.0, 167 mM NaCl, protease inhibitor cocktail - Roche) and precleared with 10 ul 50% protein A Sepharose slurry containing 1mg/ml bovine serum albumine (BSA) for 1h agitation at 4degC. Precleared chromatin was incubated with 2 ug antibody overnight at 4degC. All subsequent ChIP steps were performed as described (Rietveld, L.E., Caldenhoven, E. & Stunnenberg, H.G. In vivo repression of an erythroid-specific gene by distinct corepressor complexes. Embo J 21, 1389-97, 2002). We used the following antibodies for ChIP analyses: anti-H3K27me3 - #07-449 from Upstate; anti-H3K4me3 - ab8580-50 from Abcam; and anti-IgG - 011-0000-003 from Jackson Immuno Research. The data were normalized to a parallel ChIP with anti-H3 antibody (ab1797-100 from Abcam), to correct for differences in nucleosome density. The ChIP method was and adaptation of a previously published protocol (Rietveld, L.E., Caldenhoven, E. & Stunnenberg, H.G. In vivo repression of an erythroid-specific gene by distinct corepressor complexes. Embo J 21, 1389-97, 2002). Approximately 2x108 Kc cells were cross-linked by the addition of formaldehyde (1% final concentration). The reaction was quenched with 125 mM Glycine and the cells were washed twice with PBS. Subsequently, the cells were incubated 5� on ice in 1.5 ml lysis buffer (1% v/v SDS, 10 mM EDTA pH8.0, 50 mM Tris-HCl pH8.0, protease inhibitor cocktail - Roche). The cell lysates were sonicated and centrifuged to remove cell debris. We used the equivalent of 1 x 10E6 cells for one immunoprecipitation. Chromatin samples were diluted 10-fold in IP buffer (1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8.0, 167 mM NaCl, protease inhibitor cocktail - Roche) and precleared with 10 ul 50% protein A Sepharose slurry containing 1mg/ml bovine serum albumine (BSA) for 1h agitation at 4degC. Precleared chromatin was incubated with 2 ug antibody overnight at 4degC. All subsequent ChIP steps were performed as described (Rietveld, L.E., Caldenhoven, E. & Stunnenberg, H.G. In vivo repression of an erythroid-specific gene by distinct corepressor complexes. Embo J 21, 1389-97, 2002). We used the following antibodies for ChIP analyses: anti-H3K27me3 - #07-449 from Upstate; anti-H3K4me3 - ab8580-50 from Abcam; and anti-IgG - 011-0000-003 from Jackson Immuno Research. The data were normalized to a parallel ChIP with anti-H3 antibody (ab1797-100 from Abcam), to correct for differences in nucleosome density.
Label
Cy5
Label protocol
For the microarray analysis, immunoprecipitated DNA fragments were amplified using linker-modified PCR (LM-PCR). Randomly sheared DNA fragments were made blunt using T4 DNA polymerase for 15 min at 16�C in 10 mM Tris-HCl pH8.0, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1 mM dNTPs. Adapter ligation, PCR amplification, labeling and hybridization were carried out according to the DamID protocol (Greil, F. et al. Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev 17, 2825-38, 2003) For the microarray analysis, immunoprecipitated DNA fragments were amplified using linker-modified PCR (LM-PCR). Randomly sheared DNA fragments were made blunt using T4 DNA polymerase for 15 min at 16�C in 10 mM Tris-HCl pH8.0, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, and 0.1 mM dNTPs. Adapter ligation, PCR amplification, labeling and hybridization were carried out according to the DamID protocol (Greil, F. et al. Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev 17, 2825-38, 2003)
Drosophila Kc cells were grown in BPYE medium (Shields and Sang M3 Insect medium, Sigma, supplemented with 25% w/v bactopeptone, 20% w/v yeast extract, 5% heat inactivated fetal calf serum, all Gibco) at 23degC
Extracted molecule
genomic DNA
Label
Cy3
Description
Embryonic Drosophila cells, Kc167 cells
Data processing
After removal of low quality, redundant or ambiguous proves, hybridization data of 9,994 probes was used for analyses, all measured ratios were log base 2-transformed and normalized to the median value of the entire array
