|
| Status |
Public on Sep 09, 2014 |
| Title |
MN_Scerevisiae_rpd3_G1 |
| Sample type |
SRA |
| |
|
| Source name |
MN_Scerevisiae_rpd3_G1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: W303-1a rpd3Δ::TRP1RFA1::PK(9x)-HISV ura3::URA3/GDP-TK(7X)
culture type: G1 arrest
|
| Growth protocol |
Yeast cultures were grown in YP medium (1% yeast extract, 2% bacto peptone) supplemented with 2% glucose (YPD) at 23ºC to a final concentration of 0.7× 107 cells/ml. Cells were synchronized in G1 phase by adding 5 μg/ml α factor (αF) for one generation time and released into fresh medium in the presence of 0.2M hydroxyurea for one hour.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
200 ml cultures at 1x107 cells/ml were collected in G1 and at 60 minutes in S-phase in the presence of 0.2 M HU for preparation of mononucleosomal DNA. Cells were treated with 10 mg of Zymolyase 20T during 10 minutes at 30 ºC to induce spheroplasts formation. Mononucleosomal fragments were generated by digesting DNA with 600 or 450 units/ml (WT and rpd3 strains respectively) of micrococcal nuclease (MNAse) at 37 ºC during 10 minutes. The amount of MNase was optimized experimentally for each strain to generate a 80:20 ratio of mononucleosomes to dinucleosomes, as described (Lantermann et al. 2010). Mononucleosomal DNA was recovered from 1.5% agarose gels and sequenced in an Illumina Genome Analyzer IIx using the single-end sequencing protocol.
Mononucleosomal DNA libraries were prepared for sequencing using standard Illumina protocols
|
| |
|
| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Mononucleosomal DNA
|
| Data processing |
Alignment: bowtie 0.12.7
The smoothed signal generated by using the multilevel 1-D biorthogonal wavelet decomposition/reconstruction tool implemented in the Python pywavelets library, is used to calculate the average spacing between boundary peaks for individual nucleosomes.
The resulting combined profile was wavelet-smoothed to generate the final nucleosome positioning profile.
We used the R64-1-1 assembly of the S. cerevisiae genome, available at: http://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/assembly/285498/ , for the alignment.
genome build: R64-1-1
|
| |
|
| Submission date |
Jan 24, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Enrique Vazquez de Luis |
| E-mail(s) |
quiquevzquez@gmail.com
|
| Organization name |
CNIC
|
| Lab |
Genomics Unit
|
| Street address |
Melchor Fernandez Almagro, 3
|
| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13272 |
| Series (1) |
| GSE54377 |
Different nucleosomal landscapes at early and late replicating origins in Saccharomyces cerevisiae |
|
| Relations |
| BioSample |
SAMN02595911 |
| SRA |
SRX448776 |
