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| Status |
Public on Dec 12, 2011 |
| Title |
S2 MI2 RNAi MI2 ChIP-chip |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
S2 MI2 RNAi MI2 ChIP DNA
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: S2 cells
antibody: MI2 rabbit polyclonal (Reddy, B.A., Bajpe, P.K., Bassett, A., Moshkin, Y.M., Kozhevnikova, E., Bezstarosti, K., Demmers, J.A., Travers, A.A., and Verrijzer, C.P. (2010). Drosophila Transcription Factor Tramtrack69 Binds MEP1 to Recruit the Chromatin Remodeler NuRD. Molecular and cellular biology)
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| 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
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| 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.
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| Label |
biotin
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| Label protocol |
according to the manufacturer protocols
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| Channel 2 |
| Source name |
Input/Mock-ChIP DNA
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: S2 cells
antibody: Mock ChIPs were performed with preimmune serum (preimmune) or protein A beads (protA)
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| 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
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| Hybridization protocol |
according to the manufacturer protocols
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| Scan protocol |
according to the manufacturer protocols
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| Description |
MI2 RNAi MI2 ChIP S2 cells Biological Rep 1-2
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]
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| Phone |
0031-10-7043335
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| Organization name |
Erasmus University Medical Center
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| 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 |
| GSM802086_S2_MI2_RNAi_MI2_1.CEL.gz |
24.2 Mb |
(ftp)(http) |
CEL |
| GSM802086_S2_MI2_RNAi_MI2_2.CEL.gz |
27.6 Mb |
(ftp)(http) |
CEL |
| GSM802086_S2_MI2_RNAi_MI2_raw.bar.gz |
17.4 Mb |
(ftp)(http) |
BAR |
| GSM802086_S2_MI2_RNAi_MI2_raw.sgr.gz |
32.3 Mb |
(ftp)(http) |
SGR |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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