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Sample GSM365614 Query DataSets for GSM365614
Status Public on Jan 31, 2009
Title Ctr9_ChIP
Sample type genomic
 
Channel 1
Source name GFP ChIP DNA from BAC-transgenic mouse ESCs
Organism Mus musculus
Characteristics Amplification method: random primer / adaptor mediated PCR (Affymetrix)
Antibody: GFP goat polyclonal
Treatment protocol 1X107 Lap-tagged Ctr9 BAC-transgenic ES cells were crosslinked with 1% (v/v) formaldehyde for 10 min at room temperature. Formaldehyde was then inactivated by the addition of 125 mM glycine. Cell extracts were sonicated into approximately 500-nt chromatin fragments using a Branson 450-D sonicator.
Growth protocol LAP-tagged Ctr9 BAC-transgenic ES cells were cultured on gelatin-coated plates in Glasgow Minimum Essential Medium (Sigma) supplemented with 10% FBS (Pan biotech), 2.2 mM L-Glutamine, 1 mM Sodium Pyruvate, 50 μM 2-mercaptoethanol, 1x NEAA (Invitrogen), and LIF (generated in house). ES cells were trypsinized and splitted every 2 days, and the medium was changed daily.
Extracted molecule genomic DNA
Extraction protocol Chromatin extracts containing DNA fragments were immunoprecipitated using a polyclonal goat anti-GFP antibody (MPI-CBG, protein facility).mmunoprecipiated protein-DNA complexes were elutated from protein G sepharose (Fastflow GE healthcare) using 200 μl elution buffer (1% SDS, 0.1M NaHCO3), and incubated at 65 °C overnight to reverse the 5 crosslinks. Elutions were treated with RNase A and proteinase K, and purified with a PCR DNA purification kit (Qiagen).
Label biotin
Label protocol ChIPed DNA were amplified using random primer / adaptor mediated PCR (Affymetrix), fragmented, and labeled using GeneChip WT Double-stranded DNA terminal Labeling Kit (Affymetrix).
 
Channel 2
Source name Input DNA from BAC-transgenic mouse ESCs
Organism Mus musculus
Characteristics Amplification method: random primer / adaptor mediated PCR (Affymetrix)
Input DNA
Treatment protocol 1X107 Lap-tagged Ctr9 BAC-transgenic ES cells were crosslinked with 1% (v/v) formaldehyde for 10 min at room temperature. Formaldehyde was then inactivated by the addition of 125 mM glycine. Cell extracts were sonicated into approximately 500-nt chromatin fragments using a Branson 450-D sonicator.
Growth protocol LAP-tagged Ctr9 BAC-transgenic ES cells were cultured on gelatin-coated plates in Glasgow Minimum Essential Medium (Sigma) supplemented with 10% FBS (Pan biotech), 2.2 mM L-Glutamine, 1 mM Sodium Pyruvate, 50 μM 2-mercaptoethanol, 1x NEAA (Invitrogen), and LIF (generated in house). ES cells were trypsinized and splitted every 2 days, and the medium was changed daily.
Extracted molecule genomic DNA
Extraction protocol Chromatin extracts containing DNA fragments were immunoprecipitated using a polyclonal goat anti-GFP antibody (MPI-CBG, protein facility).mmunoprecipiated protein-DNA complexes were elutated from protein G sepharose (Fastflow GE healthcare) using 200 μl elution buffer (1% SDS, 0.1M NaHCO3), and incubated at 65 °C overnight to reverse the 5 crosslinks. Elutions were treated with RNase A and proteinase K, and purified with a PCR DNA purification kit (Qiagen).
Label biotin
Label protocol ChIPed DNA were amplified using random primer / adaptor mediated PCR (Affymetrix), fragmented, and labeled using GeneChip WT Double-stranded DNA terminal Labeling Kit (Affymetrix).
 
 
Hybridization protocol ChIP samples were hybridized to Mouse promoter 1.0R arrays (Affymetrix).
Scan protocol Arrays were scanned on an Affymetrix Scanner 3000 7G.
Description Ctr9 ChIP, Technical Rep. 1-3, Promoter
Data processing Data was processed with Partek Genomic Suite.The following steps were applied to the raw intensities: probe sequences adjustment, RMA background correction and quantile normalization. Each probe intensity was reduced by a baseline intensity, which was defined as an average of three control replicates.

Ctr9_binding_mm8.bed contains a list of Ctr9 binding sites identified by the data processing algorithm. Each row of the file contains three columns: the name of the chromosome, the starting position and the ending position of a binding site. Positions are based on the mm8 mouse genome assembly.
 
Submission date Jan 30, 2009
Last update date Jan 30, 2009
Contact name Li Ding
E-mail(s) ding@mpi-cbg.de
Organization name MPI-CBG
Lab Buchholz Lab
Street address Pfotenhauerstr. 108
City Dresden
ZIP/Postal code 01307
Country Germany
 
Platform ID GPL5811
Series (1)
GSE14654 Ctr9 Promoter Region Binding

Supplementary file Size Download File type/resource
GSM365614_C_Mock-1.CEL.gz 32.8 Mb (ftp)(http) CEL
GSM365614_C_Mock-2.CEL.gz 32.4 Mb (ftp)(http) CEL
GSM365614_C_Mock-3.CEL.gz 32.1 Mb (ftp)(http) CEL
GSM365614_Ctr9_binding_mm8.bed.gz 15.7 Kb (ftp)(http) BED
GSM365614_T_Sh2bp1-1.CEL.gz 33.0 Mb (ftp)(http) CEL
GSM365614_T_Sh2bp1-2.CEL.gz 32.9 Mb (ftp)(http) CEL
GSM365614_T_Sh2bp1-3.CEL.gz 32.4 Mb (ftp)(http) CEL
Processed data provided as supplementary file

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