 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 22, 2013 |
| Title |
ctrl_WT_del_rep01 |
| Sample type |
SRA |
| |
|
| Source name |
Wild-type control for deletion strains
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741, MATa his3-del1 leu2-del0 met15-del0 ura3-del0
genotype/variation: Wild-type control for deletion strains
|
| Growth protocol |
For deletion strain controls, 5 mL cultures were grown overnight at 30 °C in YPD. Cells were diluted to OD600 0.2/mL in 400 mL of YPD media the next morning, and grown to mid-log phase (OD600 0.8-1.0/ml) in 1L flasks at 30 °C while shaking, at which point RNA and nucleosomal DNA were isolated.
|
| 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.
|
| |
|
| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Monococcal nuclease treated chromatin profile for wild-type strain
|
| 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.
|
| |
|
| Submission date |
Mar 05, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
harm.vanbakel@mssm.edu
|
| Organization name |
Mount Sinai School of Medicine
|
| Department |
Genetics and Genomic Sciences
|
| Lab |
Bakel Lab
|
| Street address |
One Gustave L. Levy Place, Box 1498
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| 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 |
|
| Relations |
| SRA |
SRX248506 |
| BioSample |
SAMN01974804 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1093420_ctrl_WT_del_rep01.midpoints.wig.gz |
32.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
