|
| Status |
Public on Mar 16, 2010 |
| Title |
transcript levels in Kc and HP1 knockdown cells, sch20070412dtr |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
random-primed cDNA from total RNA of Kc cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
female, embryonic origin
|
| Treatment protocol |
Double stranded RNA (dsRNA) was prepared from a PCR product spanning the entire HP1 coding region, generated with primers containing a T7 RNA Polymerase binding site using the MEGASCRIPT T7 In Vitro Transcription Kit (Ambion, Cat.No. AM1334). The RNA was purified and heated to 70○C for 10 minutes and slowly cooled down to room temperature for about 30 minutes to enhance annealing. 50μg of dsRNA was added to 106 cells every 2nd day and 8 days after initial addition of dsRNA cells were harvested and the efficiency of HP1 reduction was estimated by western blot analysis using a monoclonal α-HP1 antibody.
|
| Growth protocol |
Drosophila Kc cells were kept in HyQ-SFX (Hyclone).
|
| Extracted molecule |
total RNA |
| 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. RNA was isolated 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). ChIP for H3K27me3 and H3K9me2 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 |
random-primed cDNA from total RNA from Cl8 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
female, embryonic origin
cell type: total RNA from HP1 knockdown cells
|
| Treatment protocol |
Double stranded RNA (dsRNA) was prepared from a PCR product spanning the entire HP1 coding region, generated with primers containing a T7 RNA Polymerase binding site using the MEGASCRIPT T7 In Vitro Transcription Kit (Ambion, Cat.No. AM1334). The RNA was purified and heated to 70○C for 10 minutes and slowly cooled down to room temperature for about 30 minutes to enhance annealing. 50μg of dsRNA was added to 106 cells every 2nd day and 8 days after initial addition of dsRNA cells were harvested and the efficiency of HP1 reduction was estimated by western blot analysis using a monoclonal α-HP1 antibody.
|
| Growth protocol |
Drosophila Kc cells were kept in HyQ-SFX (Hyclone).
|
| Extracted molecule |
total RNA |
| 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. RNA was isolated 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). ChIP for H3K27me3 and H3K9me2 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 |
Total RNA was measured in Kc cells with and without HP1 knockdown.
|
| 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 analysis, and 200bp for H3K27me3, H3K9me2, 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.
HMM_based_differentially replicating regions_Kc-HP1.txt: Hidden Markov Model segmentation of replication timing differences between Kc and HP1 kd cells (HMM switch.type, difference between Comparison 1 and Comparison 1 of GSE13328), including a cutoff for strong replication timing differences (significant replication timing switch). Transcription differences (Comparison 2), replication timing (Comparison 1 and Comparison 1 of GSE13328), HP1 binding, H3K27me3 (Comparison 3), H3K9me2 (Comparison 4), numer of repeats, and repeat density (percentage of sequence covered by repeats) within HMM based regions are included (as average signal within HMM based region).
|
| |
|
| Submission date |
Sep 14, 2009 |
| Last update date |
Mar 16, 2010 |
| Contact name |
Michaela Schwaiger |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Vienna
|
| Lab |
Technau
|
| Street address |
Althanstr. 14
|
| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL5919 |
| Series (1) |
| GSE18092 |
Heterochromatin protein 1 (HP1) modulates replication timing of Drosophila heterochromatin |
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