|
| Status |
Public on Aug 31, 2010 |
| Title |
HSF1 SOM-rep2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Amplified HSF1 ChIP DNA from mouse wild-type testis labeled with Cy3.
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Wild-type (C57BL/6J)
age: 60 d
tissue: testis
sample type: Wild-type (C57BL/6J) adult (60 days old) mice testis used as material
strain: C57BL/6J
antibody: HSF1 polyclonal antibody
antibody vendor: Stressgen
antibody catalog #: SPA-901
|
| Treatment protocol |
Adult (60 days old) wild-type mice were used for isolation of testes
|
| Growth protocol |
Pathogen-free mice were housed under controlled environmental conditions and fed with complete pelleted chow and allowed tap water. The mice were killed by CO2 asphyxiation. All mice were handled in accordance with the institutional animal care policies
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Isolated testes were cross-linked with a final concentration of 1% formaldehyde. Quenching was performed with 125 mM glycine. Testes were lysed in 4 ml lysis buffer. Chromatin samples were sonicated with Bioruptor (Diagenode) to an approximate size of 100-500 bp. Immunoprecipitation was performed after preclearing with 50% slurry of
protein G-coated Sepharose beads (Amersham Biosciences), at 4C over night. HSF1 antibody was used for immunoprecipitation and an input sample was obtained after the immunoprecipitation. Immunocomplexes were washed eight times. Cross-links were reversed by incubation of samples over night at 65C and DNA was purified and resuspended in water.
DNA amplification of material obtained from three biological replicates, needed for microarray hybridizations. Whole ChIP samples and 20 ng of the input samples were used for the ligation-mediated PCR (LM-PCR). The DNA was blunted using dNTP mix (Promega) and T4 DNA polymerase (New England Biolabs), purified and dissolved in water.
Purified DNA was ligated using T4 DNA ligase (New England Biolabs) and annealed with linkers made from HPLC-purified oligos; oligo 1: 5’-gcg gtg acc ggg aga tct gaa ttc-3’ and oligo 2: 5’-gaa ttc aga tc-3’. DNA was purified again and dissolved in water. LM-PCR was performed using Taq Polymerase (New England Biolabs) and Pfu Polymerase (Stratagen).
|
| Label |
Cy3
|
| Label protocol |
For NimbleGen arrays, ChIP DNA prepared from testes was directly labeled by klenow random priming with Cy5 or Cy3 nonamers per manufacturer's protocol: http://www.nimblegen.com/products/lit/lit.html
|
| |
|
| Channel 2 |
| Source name |
Amplified Input DNA from mouse wild-type testis labeled with Cy5.
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Wild-type (C57BL/6J)
sample type: Wild-type (C57BL/6J) adult (60 days old) mice testis used as material
strain: C57BL/6J
chip antibody: none, input DNA
age: 60 d
tissue: testis
|
| Treatment protocol |
Adult (60 days old) wild-type mice were used for isolation of testes
|
| Growth protocol |
Pathogen-free mice were housed under controlled environmental conditions and fed with complete pelleted chow and allowed tap water. The mice were killed by CO2 asphyxiation. All mice were handled in accordance with the institutional animal care policies o
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Isolated testes were cross-linked with a final concentration of 1% formaldehyde. Quenching was performed with 125 mM glycine. Testes were lysed in 4 ml lysis buffer. Chromatin samples were sonicated with Bioruptor (Diagenode) to an approximate size of 100-500 bp. Immunoprecipitation was performed after preclearing with 50% slurry of
protein G-coated Sepharose beads (Amersham Biosciences), at 4C over night. HSF1 antibody was used for immunoprecipitation and an input sample was obtained after the immunoprecipitation. Immunocomplexes were washed eight times. Cross-links were reversed by incubation of samples over night at 65C and DNA was purified and resuspended in water.
DNA amplification of material obtained from three biological replicates, needed for microarray hybridizations. Whole ChIP samples and 20 ng of the input samples were used for the ligation-mediated PCR (LM-PCR). The DNA was blunted using dNTP mix (Promega) and T4 DNA polymerase (New England Biolabs), purified and dissolved in water.
Purified DNA was ligated using T4 DNA ligase (New England Biolabs) and annealed with linkers made from HPLC-purified oligos; oligo 1: 5’-gcg gtg acc ggg aga tct gaa ttc-3’ and oligo 2: 5’-gaa ttc aga tc-3’. DNA was purified again and dissolved in water. LM-PCR was performed using Taq Polymerase (New England Biolabs) and Pfu Polymerase (Stratagen).
|
| Label |
Cy5
|
| Label protocol |
For NimbleGen arrays, ChIP DNA prepared from testes was directly labeled by klenow random priming with Cy5 or Cy3 nonamers per manufacturer's protocol: http://www.nimblegen.com/products/lit/lit.html
|
| |
|
| |
| Hybridization protocol |
The labeled ChIP DNA was precipitated and hybridized in buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA. Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C and then washed. Hybridizations were performed per manufacturer's protocols
http://www.nimblegen.com/products/lit/lit.html
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner per manufacturers's protocol: http://www.nimblegen.com/products/lit/lit.html
|
| Description |
Mapping of in vivo targets for HSF1 in mouse testis by using a global ChIP-chip promoter screen. A 1.5 kb promoter tiling array (NimbleGen Systems Inc.), covering ~26000 promoters of the mouse genome was used.
|
| Data processing |
The ChIP-chip data was normalized with Lowess normalization method and normalized log2 ratio (HSF1 ChIP/Input DNA) was produced for each biological replicate. The promoter values were calculated as average log2 ratios over all probes for each promoter. To identify the HSF1 target population, we used a non-arbitrary analysis method called RankProd described in Breitling et al. 2004. RankProd has been published as an R/Bioconductor package.
|
| |
|
| Submission date |
Jun 22, 2010 |
| Last update date |
Aug 31, 2010 |
| Contact name |
Lea Sistonen |
| E-mail(s) |
lea.sistonen@btk.fi
|
| Organization name |
Åbo Akademi University
|
| Department |
Biology
|
| Street address |
Tykistokatu 6 B
|
| City |
Turku |
| ZIP/Postal code |
20520 |
| Country |
Finland |
| |
|
| Platform ID |
GPL6344 |
| Series (1) |
| GSE22492 |
HSF1 regulates transcription of sex-chromosomal multicopy genes during post-meiotic repression |
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