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| Status |
Public on Dec 12, 2011 |
| Title |
S2 Mock RNAi H3 ChIP-chip |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
S2 Mock RNAi H3 ChIP DNA
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: S2 cells
antibody: H3 rabbit polyclonal (ab1791, Abcam)
|
| Treatment protocol |
Drosophila S2 cells were treated with double-stranded RNA (dsRNA) for 4 days as described (Worby, C. A., N. Simonson-Leff, and J. E. Dixon. 2001. RNA interference of gene expression (RNAi) in cultured Drosophila cells. Sci. STKE 2001:PL1). Double-stranded RNA was synthesized using an Ambion Megascript T7 kit according to the manufacturer's protocol.
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| Growth protocol |
Drosophila S2 cells were cultured in Schneider's media (Invitrogen 21720-024) supplied with 10% FBS and Penicillin/Streptomycin mixture at 25°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The following antibodies were used: anti-H3 (ab1791, Abcam), anti-H2B (raised against purified Drosophila core histones), anti-BAP111 and anti-BRM (Chalkley et al., 2008), anti-ISWI Ab14 (584-598aa), anti-ISWI Ab18 (1012-1027aa), anti-MI2 anti-MEP1 (Reddy et al., 2010) and anti-INO8016-310 (Klymenko et al., 2006). Log-phase S2 cells were fixed for 10 min with 1% formaldehyde. Fixation was stopped by 125 mM glycine, cells were washed with PBS and resuspended in ice-cold L buffer (1% sodium dodecyl sulfate, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 0.5 mM phenylmethylsulfonyl fluoride - PMSF, and 100 ng/ml of leupeptin and aprotinin). Cross-linked chromatin was sonicated to an apparent length of ~300-500 bp (corresponding to ~200-300 bp of free DNA). For Drosophila chromatin, 2 g of embryos, or larvae, were homogenized and fixed for 15’ in H buffer (30 mM KCl, 15 mM NaCl, 2 mM MgCl2, 7.5 mM HEPES pH 7.6, 0.5% Triton X-100 and 0.5 mM DTT) containing 2% formaldehyde. After blocking with 0.125 M glycine, the lysate was filtered through miracloth. Nuclei were purified by centrifugation through buffer H, containing 0.15 M sucrose and resuspended in ice-cold buffer L and sonicated as described above. 100 mkg of cross-linked chromatin was diluted with 9 volumes of buffer D (150 mM NaCl, 20 mM Tris-HCl pH8.1, 2 mM EDTA pH8.0, 1% Triton-X100, 0.5 mM PMSF, and 100 ng/ml of leupeptin and aprotinin), pre-cleared with 10 mkl (bed volume) protein A agarose blocked with salmon sperm DNA (16-157, Upstate), incubated ON at 4°C with ~10 mkl of appropriate antibodies, followed by 1 hr incubation with 20 mkl of pre-blocked protein A agarose. For Mock ChIP, chromatin was incubated with either 10 mkl of preimmune serum (4 replicas) or directly with 20 mkl of pre-blocked protein A agarose (6 replicas). Beads were washed 5x with buffer W (20 mM Tris-HCl pH 8.1, 2 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100, 0.5 mM PMSF, and 100 ng/ml of leupeptin and aprotinin) containing 150 mM NaCl, and 1 x with buffer W/500 mM NaCl. DNA was eluted with 250 mkl buffer E (1% SDS, 0.1 M NaHCO3, 500 mkg/ml Proteinase K) for 2 hrs at 37°C and overnight at 65°C. DNA was then extracted with QIAquick PCR purification kit (Qiagen Cat. 28106). For FAIRE, 100 mkg of cross-linked chromatin was diluted 9x with buffer D, phenol-chloroform extracted followed by RNase A treatment (1 mkg/ml) for 2 hours at 37°C. Isolated DNA was amplified with REPLI-g (Qiagen Cat. 150025), digested with DNase, labeled and hybridized on Affymetrix Drosophila tiling 2.0R arrays.
|
| Label |
biotin
|
| Label protocol |
according to the manufacturer protocols
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| Channel 2 |
| Source name |
Input/Mock-ChIP DNA
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: S2 cells
antibody: Mock ChIPs were performed with preimmune serum (preimmune) or protein A beads (protA)
|
| Treatment protocol |
Drosophila S2 cells were treated with double-stranded RNA (dsRNA) for 4 days as described (Worby, C. A., N. Simonson-Leff, and J. E. Dixon. 2001. RNA interference of gene expression (RNAi) in cultured Drosophila cells. Sci. STKE 2001:PL1). Double-stranded RNA was synthesized using an Ambion Megascript T7 kit according to the manufacturer's protocol.
|
| Growth protocol |
Drosophila S2 cells were cultured in Schneider's media (Invitrogen 21720-024) supplied with 10% FBS and Penicillin/Streptomycin mixture at 25°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The following antibodies were used: anti-H3 (ab1791, Abcam), anti-H2B (raised against purified Drosophila core histones), anti-BAP111 and anti-BRM (Chalkley et al., 2008), anti-ISWI Ab14 (584-598aa), anti-ISWI Ab18 (1012-1027aa), anti-MI2 anti-MEP1 (Reddy et al., 2010) and anti-INO8016-310 (Klymenko et al., 2006). Log-phase S2 cells were fixed for 10 min with 1% formaldehyde. Fixation was stopped by 125 mM glycine, cells were washed with PBS and resuspended in ice-cold L buffer (1% sodium dodecyl sulfate, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 0.5 mM phenylmethylsulfonyl fluoride - PMSF, and 100 ng/ml of leupeptin and aprotinin). Cross-linked chromatin was sonicated to an apparent length of ~300-500 bp (corresponding to ~200-300 bp of free DNA). For Drosophila chromatin, 2 g of embryos, or larvae, were homogenized and fixed for 15’ in H buffer (30 mM KCl, 15 mM NaCl, 2 mM MgCl2, 7.5 mM HEPES pH 7.6, 0.5% Triton X-100 and 0.5 mM DTT) containing 2% formaldehyde. After blocking with 0.125 M glycine, the lysate was filtered through miracloth. Nuclei were purified by centrifugation through buffer H, containing 0.15 M sucrose and resuspended in ice-cold buffer L and sonicated as described above. 100 mkg of cross-linked chromatin was diluted with 9 volumes of buffer D (150 mM NaCl, 20 mM Tris-HCl pH8.1, 2 mM EDTA pH8.0, 1% Triton-X100, 0.5 mM PMSF, and 100 ng/ml of leupeptin and aprotinin), pre-cleared with 10 mkl (bed volume) protein A agarose blocked with salmon sperm DNA (16-157, Upstate), incubated ON at 4°C with ~10 mkl of appropriate antibodies, followed by 1 hr incubation with 20 mkl of pre-blocked protein A agarose. For Mock ChIP, chromatin was incubated with either 10 mkl of preimmune serum (4 replicas) or directly with 20 mkl of pre-blocked protein A agarose (6 replicas). Beads were washed 5x with buffer W (20 mM Tris-HCl pH 8.1, 2 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100, 0.5 mM PMSF, and 100 ng/ml of leupeptin and aprotinin) containing 150 mM NaCl, and 1 x with buffer W/500 mM NaCl. DNA was eluted with 250 mkl buffer E (1% SDS, 0.1 M NaHCO3, 500 mkg/ml Proteinase K) for 2 hrs at 37°C and overnight at 65°C. DNA was then extracted with QIAquick PCR purification kit (Qiagen Cat. 28106). For FAIRE, 100 mkg of cross-linked chromatin was diluted 9x with buffer D, phenol-chloroform extracted followed by RNase A treatment (1 mkg/ml) for 2 hours at 37°C. Isolated DNA was amplified with REPLI-g (Qiagen Cat. 150025), digested with DNase, labeled and hybridized on Affymetrix Drosophila tiling 2.0R arrays.
|
| Label |
biotin
|
| Label protocol |
according to the manufacturer protocols
|
| |
|
| |
| Hybridization protocol |
according to the manufacturer protocols
|
| Scan protocol |
according to the manufacturer protocols
|
| Description |
H3 ChIP Mock RNAi S2 cells Biological Rep 1-3
Test CEL files linked below. Control CEL files listed below are linked to Series record.
Control CEL file: S2_input_1.CEL
Control CEL file: S2_input_2.CEL
Control CEL file: S2_input_3.CEL
Control CEL file: S2_input_4.CEL
Control CEL file: S2_input_5.CEL
Control CEL file: S2_mock_preimmune_1.CEL
Control CEL file: S2_mock_preimmune_2.CEL
Control CEL file: S2_mock_preimmune_3.CEL
Control CEL file: S2_mock_preimmune_4.CEL
Control CEL file: S2_mock_protA_1.CEL
Control CEL file: S2_mock_protA_2.CEL
Control CEL file: S2_mock_protA_3.CEL
Control CEL file: S2_mock_protA_4.CEL
Control CEL file: S2_mock_protA_5.CEL
Control CEL file: S2_mock_protA_6.CEL
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| Data processing |
Raw ChIP-chip and FAIRE-chip hybridization intensities were analyzed using R and R/Bioconductor packages. In brief, genome-wide ChIP-chip enrichment was estimated as following: 1) log2-scaled hybridization intensities from the input genomic DNA (5 replicas) were normalized by quantile normalization using Bioconductor package preprocessCore and averaged. 2) log2 – scaled ratios of ChIP hybridization intensities to averaged input were taken and quantile normalized independently for each antibodies giving log2(ChIP/Input) values. 3) The same was done for the Mock ChIP hybridization intensities giving log2(Mock/Input) values. 4) Averaged log2(ChIP/Input) and log2(Mock/Input) ratios were scaled by the median and the log2(Mock/Input)average ratio was subtracted from the log2(ChIP/Input)average ratio giving ChIP-chip enrichment score. For FAIRE-chip, enrichment score was calculated as averaged log2(FAIRE/Input) ratio, as this assay does not rely on antibodies. For histone H3 and H2B ChIP-chip and FAIRE-chip, the resulting enrichment scores were smoothened by running mean on the window of 250 bp. For FAIRE-chip, the results were centered by the mean. Binding sites of ATP-dependent chromatin remodelers were selected based on false discovery rate (FDR) estimation using modified procedure described by (Simonis et al., 2006) as following: 1) ChIP-chip enrichment scores were randomly permutated and a running mean with a window of 500 bp was applied. This provides a "null" distribution for FDR estimation 2) FDR was set at < 0.05 by taking the value from the "null" distribution above 95% this distribution. 3) Finally, a running mean with a window of 500 bp was applied to original data and signals (peaks) above the FDR threshold were taken. In addition, we discarded the peaks represented by less than 5 probes. For each remodeler two different antibodies were used and only sites which met the threshold for both antibodies were considered as positive remodeler-bound regions. The INO80 binding sites were selected at FDR <0.01. Remodeler binding sites were derived by intersecting the sets of significant ChIP-chip peaks for each antibody (see the additional results files: (P)BAP_sites.bed, NURD_sites.bed, INO80_sites.bed, ISWI_sites.bed files, which are linked as supplementary files on the Series record)
Results file descriptions:
1) raw.bar files contain ChIP.score calculated for each antibody.
2) raw.sgr files contain chromosome, position and ChIP.score.
3) bed files contain binding sites selected at false discovery rate (FDR) of < 0.05 per individual antibody against remodelers subunits. The INO80 sites were selected at FDR < 0.01.
4) Files (P)BAP_sites.bed, NURD_sites.bed, ISWI_sites.bed contain remodeler binding sites, which were derived by intersecting the sets of significant ChIP-chip peaks for each antibody. INO80_sites.bed contains INO80 sites filtered at FDR < 0.01. These bed files are linked as supplementary files on the Series record.
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| Submission date |
Sep 27, 2011 |
| Last update date |
Dec 12, 2011 |
| Contact name |
Yuri Moshkin |
| E-mail(s) |
[email protected]
|
| Phone |
0031-10-7043335
|
| Organization name |
Erasmus University Medical Center
|
| Street address |
Dr. Molewaterplein 50
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| City |
Rotterdam |
| ZIP/Postal code |
3015 GE |
| Country |
Netherlands |
| |
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| Platform ID |
GPL6629 |
| Series (1) |
| GSE32404 |
Remodelers organize cellular chromatin by counteracting intrinsic histone-DNA sequence preferences in a class-specific manner |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM802098_S2_H3_Mocki_raw.bar.gz |
17.4 Mb |
(ftp)(http) |
BAR |
| GSM802098_S2_H3_Mocki_raw.sgr.gz |
32.2 Mb |
(ftp)(http) |
SGR |
| GSM802098_S2_Mock_RNAi_H3_1.CEL.gz |
26.9 Mb |
(ftp)(http) |
CEL |
| GSM802098_S2_Mock_RNAi_H3_2.CEL.gz |
26.6 Mb |
(ftp)(http) |
CEL |
| GSM802098_S2_Mock_RNAi_H3_3.CEL.gz |
28.0 Mb |
(ftp)(http) |
CEL |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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