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Sample GSM444365 Query DataSets for GSM444365
Status Public on Oct 30, 2018
Title SRF_ChIP
Sample type genomic
 
Channel 1
Source name SRF ChIP DNA from E11.5 heart
Organism Mus musculus
Characteristics organ: heart
tissue: cardiac
developmental stage: E11.5
antibody: SRF
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).
 
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).
 
 
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.
Scan protocol Arrays were scanned using the GeneChip Scanner 3000 7G.
Description SRF ChIP Biological Rep 1-3
Two of the raw data files (GSM444365_CS21.CEL and GSM444365_CS26.CEL) are identical to those (GSM444366_CS21.CEL and GSM444366_CS26.CEL) for sample GSM444366.
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.
 
Submission date Aug 25, 2009
Last update date Oct 30, 2018
Contact name Timothy J Mohun
E-mail(s) tmohun@nimr.mrc.ac.uk
Phone +44 (0)208 816 2522
Fax +44 (0)208 816 2009
Organization name MRC National Institute for Medical Research
Department Developmental Biology Division
Lab Mohun Lab
Street address The Ridgeway, Mill Hill,
City London
ZIP/Postal code NW7 1AA,
Country United Kingdom
 
Platform ID GPL5811
Series (1)
GSE17807 Genome-wide binding of Nkx2-5

Supplementary file Size Download File type/resource
GSM444365_CS18_input.CEL.gz 14.3 Mb (ftp)(http) CEL
GSM444365_CS20_test.CEL.gz 14.2 Mb (ftp)(http) CEL
GSM444365_CS21_input.CEL.gz 15.5 Mb (ftp)(http) CEL
GSM444365_CS23_test.CEL.gz 15.5 Mb (ftp)(http) CEL
GSM444365_CS26_input.CEL.gz 15.1 Mb (ftp)(http) CEL
GSM444365_CS27_test.CEL.gz 15.0 Mb (ftp)(http) CEL
GSM444365_v52.Mm_PromPF_v02-1_NCBI35.NR.bpmap_matscore.bar.gz 24.6 Mb (ftp)(http) BAR
GSM444365_v52.bed.gz 9.3 Kb (ftp)(http) BED
Processed data provided as supplementary file

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