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| Status |
Public on May 22, 2013 |
| Title |
ctrl_WT-wRPB2_tet |
| Sample type |
SRA |
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| Source name |
Wild-type control for RPB2_tet strain
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741, MATa his3-del1 leu2-del0 met15-del0 URA3::CMV-tTA
genotype/variation: Wild-type control for RPB2_tet strain
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| Growth protocol |
For controls of Tet promoter-shutoff strains, doxycyline was added at 10μg/ml for 24 h in order to obtain down-regulation for the gene of interest. Cells were then diluted to an OD600 of 0.2 and grown in the presence of doxycycline (10μg/ml) until mid-log phase.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked by direct addition of methanol-free formaldehyde (Polysciences) to a final concentration of 2% for 30 min while shaking at 30 °C. The reaction was quenched by adding glycine to a final concentration of 125 mM for 5 min. Cells were pelleted, washed with 20 mL phosphate buffered saline solution once, and resuspended in 6 mL of [1 M sorbitol, 50 mM Tris 7.4] with freshly added 10 mM β-mercaptoethanol in a 15 mL conical tube. Zymolyase (20T, TakaRa Biotechnology Co., Ltd, Japan) was added to a final concentration of 0.25 mg/mL and cells were spheroplasted at 30 °C while gently rolling for 30 min. After zymolyase treatment, cells were pelleted and resuspended in 4 mL of [1 M sorbitol, 50 mM NaCl, 10 mM Tris 7.4, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40] with freshly added 1 mM β-mercaptoethanol and 500 μM spermidine. Spheroplasts were divided into 6 aliquots of 300 μL each and transferred into 1.5 mL Eppendorf tubes. Micrococcal nuclease (MNase; Worthington) dissolved in water at 0.1 U/μL stock was added to the tubes at concentrations of 0, 25, 50, 75, 100, and 150 U per sample. The digestion reactions were incubated at 37 °C for 45 min and reactions were stopped by adding 75 μL of [5% SDS, 50 mM EDTA]. Remaining proteins were digested by adding 3 uL of 20 mg/mL proteinase K solution (Qiagen) was added to each tube for an overnight incubation at 65 °C. DNA from each MNase aliquot was isolated by phenol/chloroform extraction, concentrated via ethanol precipitation and treated with RNaseA (Fermentas) at a final concentration of 1mg/ml for 1 hour. Samples were then separated on a 2% agarose gel and bands corresponding to ~147bp (mono-nucleosomal fragments) were gel-extracted using the QIAquick kit (Qiagen) for each of the digestions.
Mononucleosome fragments were end-repaired, followed by amplification-free adaptor ligation and size selection on a 2% agarose gel. Clusters were generated on a single-read flowcell using Illumina’s cBot, and sequenced as 2x36 nt paired-end reads using an Illumina Genome Analyzer IIx instrument.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Monococcal nuclease treated chromatin profile for wild-type strain
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| Data processing |
library strategy: Nuc-seq
Illumina Casava1.7 software was used for basecalling.
Sequenced reads were trimmed for adaptor sequence using cutadapt and low-quality sequences were trimmed using custom perl scripts. Reads were then mapped to the May 2008 SGD S. cerevisiae genome assembly using bowtie v0.12.7 with parameters --best --strata -e 140 -a -m 1 -n 2 -5 4 -3 10 –maxins 1000
The midpoint of each mapped read pair from a mononucleosomal fragment was used as an estimate of the nucleosome center position.
Genome_build: Saccharomyces Genome Database (SGD), may 2008 build
Supplementary_files_format_and_content: Processed data files are in wig format and contain the mapped nucleosome midpoint positions. Scores represent the number of mapped midpoints.
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| Submission date |
Mar 05, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
harm.vanbakel@mssm.edu
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| Organization name |
Mount Sinai School of Medicine
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| Department |
Genetics and Genomic Sciences
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| Lab |
Bakel Lab
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| Street address |
One Gustave L. Levy Place, Box 1498
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL9377 |
| Series (2) |
| GSE44878 |
A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription [Nuc-Seq] |
| GSE44879 |
A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription |
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| Relations |
| SRA |
SRX248510 |
| BioSample |
SAMN01974808 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1093424_ctrl_WT-wRPB2_tet.midpoints.wig.gz |
29.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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