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Sample GSM1093420 Query DataSets for GSM1093420
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
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
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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