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Sample GSM2401516 Query DataSets for GSM2401516
Status Public on Nov 01, 2021
Title Mnase-seq H4_shutoff_t4_R2
Sample type SRA
Source name cultured cells
Organism Saccharomyces cerevisiae
Characteristics strain: UKY403
time point (hrs): 4
experiment type: Mnase-seq
Treatment protocol For time-course experiments, cells were initially inoculated in 5ml YPG liquid media and cultured at 30C. Logarithmically growing cells were then transferred and diluted into larger 1-1.5L YPG cultures for growth overnight at 30C while shaking in containers with 4x equivalent free-air volume. The following morning, mid-log phase cells (OD600 ~0.6-1.2) were collected by centrifugation, washed 2x within 5 minutes with ~250-500ml YP dextrose (YPD), and re-suspended in equivalent volume of YPD media for 0, 2, and 4 hours of continued growth at 30C.
Growth protocol H4 shutoff (UKY403) and isogenic control (UKY412) strains of S. cerevisiae were obtained from the Grunstein lab. Cells were maintained on agar plates containing YPG media (1% yeast extract, 2% peptone, 2% galactose).
Extracted molecule genomic DNA
Extraction protocol Cross-linked chromatin–DNA complexes were extracted by mechanical disruption (bead beating; 4 1 min sessions, 2 min on ice between sessions). An added level of standardization was achieved by quantifying the input of extracted chromatin loaded into each digest reaction; 1 mg total protein from whole-cell extracts per 200 ml digest reaction. Total protein concentration for whole cell extracts (chromatin extracts) was quantified using a Bradford assay (OD595). Following MNase treatment and cross-link reversal, DNA was phenol: chloroform precipitated, resuspended in 50 ml TE pH 8.0, and RNase treated. Small aliquots (20%, 10 ml) from each digest titration were then analyzed by gel electrophoresis. Gel intensity measurements for each lane were calculated using standard densitometry software provided by Biorad (Quantity OneTM) and exported to Microsoft ExcelTM for correlation analysis (Pearson). Correlation coefficients were calculated for digest titration levels no longer showing a visible di-nucleosome band (i.e. optimally digested samples) and spanning a region known to cover a size range of 0–400 bp of DNA. A 100-bp standard ladder (Qiagen) was analyzed on the same gel to calculate a relative front (i.e. separation relative to standard fragment sizes) for the migrating DNA population. ‘Matched’ digests were selected as two optimally digested samples having a correlation coefficient (r>0.9). Correlation coefficients r>0.9 were selected because these showed the most reproducible change in MNase protection data on single site real-time quantitative PCR (RT-qPCR) analysis (See Supplementary Methods section). The remaining sample from each optimal digest was column purified (Zymo Research), bypassing the need to gel excise mono-nucleosome DNA. Avoidance of gel extraction to isolate chromatin DNA at this stage is a key step to assure sampling of similar chromatin populations.
Matched DNA samples were prepared for sequencing using the standard Illumina protocol with additional care at the gel purification step to ensure that the same size range of DNA was selected.
Library strategy MNase-Seq
Library source genomic
Library selection MNase
Instrument model Illumina Genome Analyzer II
Description Mnase-seq of Histone H4 shutoff
Data processing alignment with Bowtie VN:0.12.7 parameters (bowtie --threads 32 -m 1 -S sgd_r64/SacCer3)
Genome_build: SacCer3
Supplementary_files_format_and_content: Processed data not available
Submission date Nov 23, 2016
Last update date Nov 01, 2021
Contact name Michael Buck
Phone 7168817569
Organization name SUNY at Buffalo
Department Biochemistry
Street address 701 Ellicott St
City Buffalo
State/province NY
ZIP/Postal code 14203
Country USA
Platform ID GPL9377
Series (2)
GSE90458 Nucleosomes inhibit intragenic binding of Rap1p and subsequent cryptic transcription [MNase-seq]
GSE92468 Nucleosomes inhibit intragenic binding of Rap1p and subsequent cryptic transcription
BioSample SAMN06050643
SRA SRX2369848

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not provided for this record

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