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 23C. Full length Nup98 was cloned into pNDamMyc (van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8, 2000) into which as Gateway cassette (Invitrogen) had been inserted in frame. Plasmid was transfected into Kc cells as described (Henikoff, S., Ahmad, K., Platero, J.S. & van Steensel, B. Heterochromatic deposition of centromeric histone H3-like proteins. Proc Natl Acad Sci U S A 97, 716-21, 2000).
Extracted molecule
genomic DNA
Extraction protocol
Sample extraction was performed as described (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)
Label
Cy3, Cy5
Label protocol
Sample labeling was performed as described (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)
Channel 2
Source name
Embryonic Drosophila cells, Kc167 cells, expressing Dam
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 23°C. Plasmid pNDamMyc (van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat Biotechnol 18, 424-8, 2000) was transfected into Kc cells as described (Henikoff, S., Ahmad, K., Platero, J.S. & van Steensel, B. Heterochromatic deposition of centromeric histone H3-like proteins. Proc Natl Acad Sci U S A 97, 716-21, 2000).
Extracted molecule
genomic DNA
Extraction protocol
Sample extraction was performed as described (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)
Label
Cy3, Cy5
Label protocol
Sample labeling was performed as described (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)
Hybridization protocol
Samples were hybridized to the arrays in a volume of approximately 30 ul (5 mM TRIS, 0.5 mM EDTA, 3X SSC). The following competitors were added: 25 ug DpnI-digested Dam-fusion plasmid DNA, 100 ug yeast tRNA, 20 ug poly (dA-dT). Arrays were washed in four subsequent steps. The respective washing buffers were: 1X SSC/0.03% SDS (arrays were submerged once), 1XSSC (arrays were submerged 15 times), 0.2XSSC (20 minutes), 0.05XSSC (10 minutes).
Scan protocol
Microarrays are scanned in a DNA Microarray Scanner (Model G2505B, Serial number US22502518) from Agilent Technologies, which uses Scan Control software (Version A.6.11). Slides were first placed in a slide holder before putting it in the Scanner Carousel, which was installed in the scanner. Slides were scanned at a variable PMT (0-100%) for each channel (Red or Green), a variable resolution (5 or 10 um per pixel) and a suitable scan area.
Scan TIFF images were processed with ImaGene 6.0.1 software (BioDiscovery) using automated grid placement with manual control and automated spot adjustment. Measurements were extracted as median spot intensities minus median background intensities. After removal of low quality (ImaGene flags), redundant or ambiguous probes, hybridization data of 9,990 probes was used for analyses, all measured ratios were log base 2-transformed and normalized within arrays to the median values using "printtiploess" and between arrays using "Aquantile" and averaged using "lmFit" using the limma software package (version 3.2.1)(Smyth, G. K. (2004). Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, No. 1, Article 3.).
