|
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.
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|
|
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
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Submission date |
Nov 23, 2016 |
Last update date |
Nov 01, 2021 |
Contact name |
Michael Buck |
E-mail(s) |
mjbuck@buffalo.edu
|
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 |
|
Relations |
BioSample |
SAMN06050643 |
SRA |
SRX2369848 |