NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM452295 Query DataSets for GSM452295
Status Public on Mar 16, 2010
Title H3K27me3 in Kc cells, sch20080807dtr2
Sample type genomic
 
Channel 1
Source name 3K27-ChIP DNA
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 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. 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 Input DNA
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 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. 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 Tri-methylation of Lysine 27 of Histone H3 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 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) 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 GPL6629
Series (1)
GSE18092 Heterochromatin protein 1 (HP1) modulates replication timing of Drosophila heterochromatin

Supplementary file Size Download File type/resource
GSM452295_3K27-Kc.txt.gz 13.0 Mb (ftp)(http) TXT
GSM452295_sch20080514dtr2_01_F_Kc_Input_15.1.08_212.CEL.gz 29.7 Mb (ftp)(http) CEL
GSM452295_sch20080807dtr2_01_WT_Kc_H3K27me3_15.1.08-5_236.CEL.gz 28.6 Mb (ftp)(http) CEL
GSM452295_sch20080807dtr2_02_WT_Kc_H3K27me3_14.2.08-10_236.CEL.gz 29.2 Mb (ftp)(http) CEL
GSM452295_sch20080807dtr2_03_WT_Kc_Input_14.2.08-4_236.CEL.gz 28.3 Mb (ftp)(http) CEL
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap
External link. Please review our privacy policy.