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| Status |
Public on Oct 30, 2018 |
| Title |
RNAPII_ChIP |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
RNAPII ChIP DNA from E11.5 heart
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| Organism |
Mus musculus |
| Characteristics |
organ: heart
tissue: cardiac
developmental stage: E11.5
antibody: RNA polymerase II
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from 20-25 mouse embryonic hearts at stage E11.5. Tissue was fixed for 3 hours at room temperature in buffer (50mM HEPES pH7.9, 1mM EDTA, 1mM EGTA, 100mM NaCl, 0.07% butryric acid) containing 1.8% formaldehyde. The tissue homogenized using an Ultra-Turrax, T25 basic (IKA-Werke), and pelleted at low speed. Samples were washed twice with ice-cold PBS with freshly added EDTA-free protease inhibitors (PI) (Roche). Samples were resuspended in 1.5ml lysis buffer (25mM Tris pH 7.5, 150mM NaCl, 1% Triton X100, 1% SDS, 2.5mM Sodium deoxycholate, PI) and transferred to RNAse-free, non-stick microfuge tubes (Ambion). Samples sonicated for 15 x 30 seconds on ice using a Branson Digital Sonicator with a 2.5mm stepped probe tip. Samples were spun at 15K using a bench top centrifuge for 15 minutes at 4ºC. The supernatant was then pre-blocked with protein A/G sepharose (Perbio), pre-treated with BSA (Biorad) and Poly (dI-dC)-poly (dI-dC) (GE Healthcare). A proportion of the sample was kept at this stage as an input control. 150μl of chromatin was diluted 10 fold in ChIP dilution buffer (16.7mM Tris pH7.5, 0.01% SDS, 1.1% Triton, 1.2mM EDTA, 167mM NaCl) and specific antibody was added and incubated overnight at 4ºC with rotation. The antibody:protein:DNA complexes were captured using pre-blocked protein A/G sepharose for 2 hours rotating at 4ºC. The beads were then spun down and washed twice in wash buffer A (10mM HEPES pH 7.6, 1mM EDTA, 0.5mM EGTA, 0.25% Triton X100) and twice with wash buffer B (10mM HEPES pH7.6, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.01% Triton X100. The beads were resuspended in TE and cross-links reversed overnight at 65ºC. Samples were adjusted to 0.1% SDS, digested with 10μg Proteinase K (Roche) at 50ºC for 3 hours and extracted twice with phenol/chloroform. Samples were ethanol precipitated and resuspended in 100μl TE. To generate sufficient material for hybridization to the Affymetrix GeneChIP® Mouse Promoter 1.0R Array, immunoprecipitated DNA from three ChIP experiments or 10ng of input DNA as control, were amplified using ligated-mediated PCR (LM-PCR). The ChIP-chip assay was performed using a minimum of three biologically independent samples.
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| Label |
biotin
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| Label protocol |
Immunoprecipitated DNA and 10ng of input genomic DNA was amplified using ligated-mediated PCR (LM_PCR) and then enzymatically fragmented and labeled with biotin using the WT Labeling Kit (Affymetrix).
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| Channel 2 |
| Source name |
Input DNA from E11.5 hearts
|
| Organism |
Mus musculus |
| Characteristics |
organ: heart
tissue: cardiac
developmental stage: E11.5
antibody: none
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was prepared from 20-25 mouse embryonic hearts at stage E11.5. Tissue was fixed for 3 hours at room temperature in buffer (50mM HEPES pH7.9, 1mM EDTA, 1mM EGTA, 100mM NaCl, 0.07% butryric acid) containing 1.8% formaldehyde. The tissue homogenized using an Ultra-Turrax, T25 basic (IKA-Werke), and pelleted at low speed. Samples were washed twice with ice-cold PBS with freshly added EDTA-free protease inhibitors (PI) (Roche). Samples were resuspended in 1.5ml lysis buffer (25mM Tris pH 7.5, 150mM NaCl, 1% Triton X100, 1% SDS, 2.5mM Sodium deoxycholate, PI) and transferred to RNAse-free, non-stick microfuge tubes (Ambion). Samples sonicated for 15 x 30 seconds on ice using a Branson Digital Sonicator with a 2.5mm stepped probe tip. Samples were spun at 15K using a bench top centrifuge for 15 minutes at 4ºC. The supernatant was then pre-blocked with protein A/G sepharose (Perbio), pre-treated with BSA (Biorad) and Poly (dI-dC)-poly (dI-dC) (GE Healthcare). A proportion of the sample was kept at this stage as an input control. 150μl of chromatin was diluted 10 fold in ChIP dilution buffer (16.7mM Tris pH7.5, 0.01% SDS, 1.1% Triton, 1.2mM EDTA, 167mM NaCl) and specific antibody was added and incubated overnight at 4ºC with rotation. The antibody:protein:DNA complexes were captured using pre-blocked protein A/G sepharose for 2 hours rotating at 4ºC. The beads were then spun down and washed twice in wash buffer A (10mM HEPES pH 7.6, 1mM EDTA, 0.5mM EGTA, 0.25% Triton X100) and twice with wash buffer B (10mM HEPES pH7.6, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.01% Triton X100. The beads were resuspended in TE and cross-links reversed overnight at 65ºC. Samples were adjusted to 0.1% SDS, digested with 10μg Proteinase K (Roche) at 50ºC for 3 hours and extracted twice with phenol/chloroform. Samples were ethanol precipitated and resuspended in 100μl TE. To generate sufficient material for hybridization to the Affymetrix GeneChIP® Mouse Promoter 1.0R Array, immunoprecipitated DNA from three ChIP experiments or 10ng of input DNA as control, were amplified using ligated-mediated PCR (LM-PCR). The ChIP-chip assay was performed using a minimum of three biologically independent samples.
|
| Label |
biotin
|
| Label protocol |
Immunoprecipitated DNA and 10ng of input genomic DNA was amplified using ligated-mediated PCR (LM_PCR) and then enzymatically fragmented and labeled with biotin using the WT Labeling Kit (Affymetrix).
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| Hybridization protocol |
5 micrograms of DNA was hybridized per array using the Affymetrix hybridization kit. The arrays were hybridized for 16hr at 45C at 60rpm using an Affymetrix hybridization oven.
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| Scan protocol |
Arrays were scanned using the GeneChip Scanner 3000 7G.
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| Description |
RNAPII ChIP Biological Rep 1-3
Two of the raw data files (GSM444366_CS21.CEL and GSM444366_CS26.CEL) are identical to those (GSM444365_CS21.CEL and GSM444365_CS26.CEL) for sample GSM444365.
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| Data processing |
The enriched transcription factor binding regions were detected by analysis of three independent biological repeats using the Model-based Analysis of Tiling-array (MAT) algorithm (Johnson et al. 2006) using a bandwidth of 300bp and a P-value cutoff of 1e-5 (.bed files)(BPMAP = Mm_PromPF_v02-1_NCBI35.NR.bpmap).
bed files were generated using the Model-based Analysis of Tiling-array (MAT) algorithm using three biological repeats of specific antibody ChIP versus three biological repeats of input.
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| Submission date |
Aug 25, 2009 |
| Last update date |
Oct 30, 2018 |
| Contact name |
Timothy J Mohun |
| E-mail(s) |
tmohun@nimr.mrc.ac.uk
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| Phone |
+44 (0)208 816 2522
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| Fax |
+44 (0)208 816 2009
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| Organization name |
MRC National Institute for Medical Research
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| Department |
Developmental Biology Division
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| Lab |
Mohun Lab
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| Street address |
The Ridgeway, Mill Hill,
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| City |
London |
| ZIP/Postal code |
NW7 1AA, |
| Country |
United Kingdom |
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| Platform ID |
GPL5811 |
| Series (1) |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM444366_CS19_test.CEL.gz |
14.3 Mb |
(ftp)(http) |
CEL |
| GSM444366_CS21_input.CEL.gz |
15.5 Mb |
(ftp)(http) |
CEL |
| GSM444366_CS22_test.CEL.gz |
15.3 Mb |
(ftp)(http) |
CEL |
| GSM444366_CS24_input.CEL.gz |
15.8 Mb |
(ftp)(http) |
CEL |
| GSM444366_CS26_input.CEL.gz |
15.1 Mb |
(ftp)(http) |
CEL |
| GSM444366_CS28_test.CEL.gz |
15.3 Mb |
(ftp)(http) |
CEL |
| GSM444366_vRNAPol.Mm_PromPF_v02-1_NCBI35.NR.bpmap_matscore.bar.gz |
24.6 Mb |
(ftp)(http) |
BAR |
| GSM444366_vRNAPol.bed.gz |
34.5 Kb |
(ftp)(http) |
BED |
| Processed data provided as supplementary file |
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