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Status
Public on Dec 01, 2017
Title
Plakoglobin ChIP 3
Sample type
genomic
Channel 1 Source name
Plakoglobin (PG) ChIP DNA from mouse keratinocytes
Organism
Mus musculus Characteristics
strain: C57BL/6J
Treatment protocol
at confluency, terminal differentiation was initiated with high calcium medium
Growth protocol
keratinocytes were grown till confluency, then four days in high calcium medium and harvested
Extracted molecule
genomic DNA
Extraction protocol
Four days after calcium switch, keratinocytes grown in 4x75cm2 flasks were washed with PBS(+) and proteins cross-linked with 2mM di-succinimidyl-glutarate(5) for 45min at RT followed by 1% formaldehyde for 15min at RT. Cells were scraped into 3ml PBS(+), centrifuged at 1,000rpm for 10min at RT and cell pellets lysed with 500μl lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.0). To shear DNA, lysates were sonicated in a Covaris S2 water bath sonicator (Covaris; Raublingen, Germany) with settings for 500bp DNA fragment length, confirmed by BioAnalyzer©. Samples were diluted 1:1 with 70mM HEPES, pH 7.5, 2.5mM NaCl, 1.5mM EDTA, 1.5% Triton-X100, 0.6% Deoxycholate (DOC) and centrifuged for 30min at 14,000rpm (Centrifuge 5417R, Eppendorf). The supernatant was pre-cleared with tRNA blocked Dynabeads® (Invitrogen Switzerland) for 2h at 4°C by rotation (Input). Immunoprecipitation was performed by rotating with 5µg antibody (PG, β-catenin (BD Biosciences)) per 1ml lysate at 4°C overnight, and 30µl Dynabeads® (pre-blocked with 1mg/ml tRNA for 1h at 4°C rotating) were added the next day for 3h at 4°C. Beads were washed twice with 1ml mixed micelle washing buffer (150mM NaCl, 100mM Tris-HCl, pH 8.1, 100mM EDTA, pH 8.0, 5% w/v sucrose, 0.02% NaN3, 1% Triton X-100, 0.2% SDS), twice with 1ml buffer 500 (0.1% DOC, 1mM EDTA, pH 8.0, 50mM HEPES, pH 7.5, 500mM NaCl, 1% Triton X-100, 0.2% NaN3), twice with 1ml LiCl buffer (0.5% DOC, 1mM EDTA, pH 8.0, 250mM LiCl, 0.5% NP-40, 10mM Tris-HCl, pH 8.0, 0.2% NaN3) and once with 1ml TE buffer. Protein-DNA complexes were eluted with 100µl elution buffer (1% SDS, 1mM NaHCO3) for 2x30min at 60°C. Samples were diluted 1:1 with TE buffer and NaCl (0.5M final concentration), reverse cross-linked overnight at 65°C followed by 100ng/µl proteinase K and 100ng/µl RNase A treatment for 1h at 55°C. A tenth volume 4M LiCl was added and proteins extracted with phenol:chloroform:isoamyl alcohol. DNA was precipitated with 100% ice cold ethanol and washed with 70% ethanol before resuspending in 50µl dH2O. The input sample was treated in the same way. The DNA amount was determined using NanoDrop
Label
Cy5
Label protocol
according to NimbleGen standart protocol, performed by NimbleGen
Channel 2 Source name
Input DNA from mouse keratinocytes
Organism
Mus musculus Characteristics
strain: C57BL/6J
Treatment protocol
at confluency, terminal differentiation was initiated with high calcium medium
Growth protocol
keratinocytes were grown till confluency, then four days in high calcium medium and harvested
Extracted molecule
genomic DNA
Extraction protocol
Four days after calcium switch, keratinocytes grown in 4x75cm2 flasks were washed with PBS(+) and proteins cross-linked with 2mM di-succinimidyl-glutarate(5) for 45min at RT followed by 1% formaldehyde for 15min at RT. Cells were scraped into 3ml PBS(+), centrifuged at 1,000rpm for 10min at RT and cell pellets lysed with 500μl lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.0). To shear DNA, lysates were sonicated in a Covaris S2 water bath sonicator (Covaris; Raublingen, Germany) with settings for 500bp DNA fragment length, confirmed by BioAnalyzer©. Samples were diluted 1:1 with 70mM HEPES, pH 7.5, 2.5mM NaCl, 1.5mM EDTA, 1.5% Triton-X100, 0.6% Deoxycholate (DOC) and centrifuged for 30min at 14,000rpm (Centrifuge 5417R, Eppendorf). The supernatant was pre-cleared with tRNA blocked Dynabeads® (Invitrogen Switzerland) for 2h at 4°C by rotation (Input). Immunoprecipitation was performed by rotating with 5µg antibody (PG, β-catenin (BD Biosciences)) per 1ml lysate at 4°C overnight, and 30µl Dynabeads® (pre-blocked with 1mg/ml tRNA for 1h at 4°C rotating) were added the next day for 3h at 4°C. Beads were washed twice with 1ml mixed micelle washing buffer (150mM NaCl, 100mM Tris-HCl, pH 8.1, 100mM EDTA, pH 8.0, 5% w/v sucrose, 0.02% NaN3, 1% Triton X-100, 0.2% SDS), twice with 1ml buffer 500 (0.1% DOC, 1mM EDTA, pH 8.0, 50mM HEPES, pH 7.5, 500mM NaCl, 1% Triton X-100, 0.2% NaN3), twice with 1ml LiCl buffer (0.5% DOC, 1mM EDTA, pH 8.0, 250mM LiCl, 0.5% NP-40, 10mM Tris-HCl, pH 8.0, 0.2% NaN3) and once with 1ml TE buffer. Protein-DNA complexes were eluted with 100µl elution buffer (1% SDS, 1mM NaHCO3) for 2x30min at 60°C. Samples were diluted 1:1 with TE buffer and NaCl (0.5M final concentration), reverse cross-linked overnight at 65°C followed by 100ng/µl proteinase K and 100ng/µl RNase A treatment for 1h at 55°C. A tenth volume 4M LiCl was added and proteins extracted with phenol:chloroform:isoamyl alcohol. DNA was precipitated with 100% ice cold ethanol and washed with 70% ethanol before resuspending in 50µl dH2O. The input sample was treated in the same way. The DNA amount was determined using NanoDrop
Label
Cy3
Label protocol
according to NimbleGen standart protocol, performed by NimbleGen
Hybridization protocol
according to NimbleGen standart protocol, performed by NimbleGen
Scan protocol
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html ).
Description
ChIP-chip mouse keratinocytes at onset of terminal differentiation using Plakoglobin antibodies
Data processing
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction.
Submission date
Apr 17, 2012
Last update date
Dec 01, 2017
Contact name
Christian Strauss
E-mail(s)
christian.strauss@vetsuisse.unibe.ch Organization name
Institute of Animal Pathology
Street address
Laengassstrasse 122
City
Berne
ZIP/Postal code
3012
Country
Switzerland
Platform ID
GPL7496 Series (1)
GSE37343 ChIP-chip from mouse keratinocytes with Plakoglobin and β-catenin
Data table header descriptions ID_REF VALUE MEAN scaled, log2 (ChIP/Input) ratio
Data table ID_REF VALUE MMUS0407S00000001
-0.182
MMUS0407S00000002
-0.034
MMUS0407S00000003
0.735
MMUS0407S00000004
0.232
MMUS0407S00000005
0.13
MMUS0407S00000006
-0.18
MMUS0407S00000007
-0.141
MMUS0407S00000008
0.175
MMUS0407S00000009
0.314
MMUS0407S00000011
0.12
MMUS0407S00000012
0.098
MMUS0407S00000013
-0.014
MMUS0407S00000015
0.08
MMUS0407S00000016
2.206
MMUS0407S00000017
0.149
MMUS0407S00000019
-0.032
MMUS0407S00000022
0.43
MMUS0407S00000024
0.396
MMUS0407S00000026
-0.175
MMUS0407S00000028
0.259
26275 617 Kbytes .Supplementary file Size Download File type/resource GSM916512_Mker_EXP3_PG_Cy3.pair.gz
6.1 Mb
(ftp) (http) PAIR
GSM916512_Mker_EXP3_PG_Cy5.pair.gz
6.1 Mb
(ftp) (http) PAIR
GSM916512_PGmeans_EXP3.txt.gz
527.2 Kb
(ftp) (http) TXT
GSM916512_PGpeaks_EXP3.txt.gz
181.1 Kb
(ftp) (http) TXT
Processed data included within Sample table Processed data provided as supplementary file
