|
| Status |
Public on Apr 11, 2009 |
| Title |
Vps75 whole genome sample 1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ChIP DNA from yeast Saccharomyces cerevisiae cells
|
| Organism |
Saccharomyces cerevisiae W303 |
| Characteristics |
genotype: leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-1
cell description: Cells expressing a C-terminally tagged version of Vps75 expressed from its own promoter (Selth et al., 2007)
strain: Vps75-HTM (Selth et al., 2007)
antibody: 9E11 anti-Myc
|
| Treatment protocol |
ChIP and hybridization protocols were performed as previously described (Selth et al. 2007; Katou et al. 2003)
|
| Growth protocol |
Cells were grown at 30C in YPD medium and cross-linked at a density of 1 x 107 cells/mL.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP analyses were performed essentially as previously described (Selth et al, 2007). Cells were grown under the conditions described above, cross-linked with formaldehyde, disrupted by bead-beating in a FastPrep 24 instrument (MP Biomedicals), and whole cell extracts sonicated to obtain 400-600 bp genomic DNA fragments. Chromatin immunoprecipitation (ChIP) was performed with 9E11 antibody (monoclonal antibody against Myc tag of Vps75) coupled to protein A-sepharose beads. Immunoprecipates were eluted and incubated overnight at 65ºC to reverse cross-links. Then, as previously described (Katou et al 2003), the immunoprecipitated genomic DNA was incubated with proteinase K, extracted twice with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RNaseA. The DNA was purified, concentrated by ethanol precipitation and amplified by PCR after random priming. 10ug of the amplified DNA was digested with DNase I and end-labelled using TdT Terminal Transferase and Biotin-N6-ddATP.
|
| Label |
DNA was labeled with biotin-N6-ddATP using terminal transferase TdT (Roche, 400U/µl)
|
| Label protocol |
DNA was labeled with biotin-N6-ddATP using terminal transferase TdT (Roche, 400U/µl).
|
| |
|
| Channel 2 |
| Source name |
Input DNA from Saccharomyces cerevisiae cells from unbound fraction
|
| Organism |
Saccharomyces cerevisiae W303 |
| Characteristics |
genotype: leu2-3,112 his3-11,15 ade2-1 ura3-1 trp1-1 can1-1
cell description: Cells expressing a C-terminally tagged version of Vps75 expressed from its own promoter (Selth et al., 2007)
strain: Vps75-HTM (Selth et al., 2007)
|
| Treatment protocol |
ChIP and hybridization protocols were performed as previously described (Selth et al. 2005; Katou et al. 2003)
|
| Growth protocol |
Cells were grown at 30C in YPD medium and cross-linked at a density of 1 x 107 cells/mL.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP analyses were performed essentially as previously described (Selth et al, 2007). Cells were grown under the conditions described above, cross-linked with formaldehyde, disrupted by bead-beating in a FastPrep 24 instrument (MP Biomedicals), and whole cell extracts sonicated to obtain 400-600 bp genomic DNA fragments. Chromatin immunoprecipitation (ChIP) was performed with 9E11 antibody (monoclonal antibody against Myc tag of Vps75) coupled to protein A-sepharose beads. Immunoprecipates were eluted and incubated overnight at 65ºC to reverse cross-links. Then, as previously described (Katou et al 2003), the immunoprecipitated genomic DNA was incubated with proteinase K, extracted twice with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RNaseA. The DNA was purified, concentrated by ethanol precipitation and amplified by PCR after random priming. 10ug of the amplified DNA was digested with DNase I and end-labelled using TdT Terminal Transferase and Biotin-N6-ddATP.
|
| Label |
DNA was labeled with biotin-N6-ddATP using terminal transferase TdT (Roche, 400U/µl)
|
| Label protocol |
DNA was labeled with biotin-N6-ddATP using terminal transferase TdT (Roche, 400U/µl).
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed as previously described (Katou et al. 2003). Samples were hybridized in buffer containing SSPE, TritonX-100, denatured salmon sperm DNA and biotin-labelled control oligonucleotide, oligo B2. Samples were denatured at 95ºC for 10 minutes and then hybridized for 16 hours at 42ºC in a hybridization oven (GeneChip Hybridization Oven 640, Affymetrix)
|
| Scan protocol |
The washing and scanning protocol (FlexMidi_euk2v3_450), provided by Affymetrix, was performed automatically on a GeneChip Fluidics Station 450. Arrays were scanned using the GeneChip Scanner 3000 7G.
|
| Description |
Genome-wide analysis of the binding pattern of Vps75 along S. cerevisiae chromosomes (First repeat)
Selth L, and Svejstrup JQ. (2007) Vps75, a new yeast member of the NAP histone chaperone family. J Biol Chem 282:12358-62.
|
| Data processing |
Probe-level signals were converted to a log base 2 scale. Each array was normalized to its median signal. Log ratios of IP-SUP were calculated for each probe.
|
| |
|
| Submission date |
Apr 08, 2009 |
| Last update date |
Jun 02, 2009 |
| Contact name |
Luke Selth |
| E-mail(s) |
luke.selth@cancer.org.uk
|
| Organization name |
Cancer Research UK
|
| Street address |
Blanch Lane
|
| City |
South Mimms |
| State/province |
Herts. |
| ZIP/Postal code |
EN6 3LD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL7249 |
| Series (1) |
| GSE15607 |
An Rtt109-independent role for Vps75 in transcription-associated nucleosome dynamics |
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