|
| Status |
Public on Feb 10, 2009 |
| Title |
H4K16 acetylation in Kc cells |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H4K16ac-CHIP DNA
|
| Organism |
Drosophila melanogaster |
| Characteristics |
female, embryonic origin
genomic DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
We sorted cells into S-phase fractions on the basis of DNA content using Fluorescent Activated Cell Sorting (FACS). We collected 60,000 cells from each fraction directly into lysis buffer. DNA was purified, sonicated, denatured and immunoprecipitated with an antibody specific for BrdU (Becton Dickinson) as described (Schubeler et al. 2002), but with two consecutive rounds of immunoprecipitation to increase specificity. olated from cells using Trizol (Invitrogen) and subsequently purified using an RNeasy kit (QIAGEN). For hybridization to Affymetrix tiling arrays, we made double-stranded cDNA by performing two rounds of cDNA synthesis using random primers and addition of 2 mM dUTPs using the GeneChip® WT Double-Stranded cDNA Synthesis Kit (Affymetrix). Nascent strand DNA in a size range of 1000 to 2000 bp was isolated from logarithmically growing Kc cells by alkaline gel electrophoresis as described (Gray et al. 2007). Genomic DNA from Kc cells in G2 phase (isolated by FACS) was used as a control that does not contain nascent strands.ChIP for H4K16 acetylation and RNA Polymerase II was carried out as described (Bell et al. 2007).To obtain sufficient target DNA for microarray hybridization, we amplified the denatured and immunoprecipitated DNA as described (Schubeler et al. 2002).
|
| Label |
Biotin
|
| Label protocol |
Samples (6-10μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| Channel 2 |
| Source name |
Input DNA
|
| Organism |
Drosophila melanogaster |
| Characteristics |
female, embryonic origin
genomic DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
We sorted cells into S-phase fractions on the basis of DNA content using Fluorescent Activated Cell Sorting (FACS). We collected 60,000 cells from each fraction directly into lysis buffer. DNA was purified, sonicated, denatured and immunoprecipitated with an antibody specific for BrdU (Becton Dickinson) as described (Schubeler et al. 2002), but with two consecutive rounds of immunoprecipitation to increase specificity. olated from cells using Trizol (Invitrogen) and subsequently purified using an RNeasy kit (QIAGEN). For hybridization to Affymetrix tiling arrays, we made double-stranded cDNA by performing two rounds of cDNA synthesis using random primers and addition of 2 mM dUTPs using the GeneChip® WT Double-Stranded cDNA Synthesis Kit (Affymetrix). Nascent strand DNA in a size range of 1000 to 2000 bp was isolated from logarithmically growing Kc cells by alkaline gel electrophoresis as described (Gray et al. 2007). Genomic DNA from Kc cells in G2 phase (isolated by FACS) was used as a control that does not contain nascent strands.ChIP for H4K16 acetylation and RNA Polymerase II was carried out as described (Bell et al. 2007).To obtain sufficient target DNA for microarray hybridization, we amplified the denatured and immunoprecipitated DNA as described (Schubeler et al. 2002).
|
| Label |
Biotin
|
| Label protocol |
Samples (6-10μg of DNA) were enzymatically fragmented and labeled using the WT Labeling Kit (Affymetrix) to add biotinylated compound to the ends of the fragments
|
| |
|
| |
| Hybridization protocol |
Approximately 6-10μg of DNA was hybridzed per array using the Affymetrix hybridization kit. Arrays were hybridized for 16 hours at 45° at 60rpm using an Affymetrix hybridization oven. Arrays were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G.
|
| Description |
Acetylation of lysine 16 of histone H4 in Kc cells.
|
| Data processing |
Tiling arrays were analyzed using MAT (Model-based Analysis of Tiling-array) software (Johnson et al. 2006). The bandwith was set to 1000bp for replication timing and nascent DNA analysis, and 300bp for H4K16ac , RNA Polymerase II and transcription data. MAT scores were extracted from the BAR files generated using the Python script ‘Bar2Wig.py’ kindly provided by Wei Li (Harvard University). Data from the Wiggle files were reformatted using Perl for subsequent analysis in R.
|
| |
|
| Submission date |
Oct 23, 2008 |
| Last update date |
Jan 04, 2012 |
| Contact name |
Michaela Schwaiger |
| E-mail(s) |
michaela.schwaiger@univie.ac.at
|
| Organization name |
University of Vienna
|
| Lab |
Technau
|
| Street address |
Althanstr. 14
|
| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL5919 |
| Series (1) |
| GSE13328 |
Chromatin structure marks cell-type and gender specific replication of the Drosophila genome |
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