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| Status |
Public on Oct 09, 2013 |
| Title |
MNase_WT_100Uml_N2 |
| Sample type |
SRA |
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| Source name |
MNase_WT_100Uml
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: FWP172
genotype/variation: wild type
mnase concentration: 100Uml
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| Treatment protocol |
cells were shifted to the non-permissive temperature of 37˚C for two hour.
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| Growth protocol |
cells from strains FWP172 (wild-type) and FWP371 (spt6-1) were grown in 200 ml of YES media to OD600=0.5.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were spheroplasted in 2 ml of CES buffer (50mM Citric acid/50mM Na2HPO pH 5.6; 40mM EDTA pH=8.0; 1.2M Sorbitol) containing 1 mg/ml Zymolyase 100T (United States Biological) and 20 mM _-mercaptoethanol for 1 hour at 30¡ C. Spheroplasts were pelleted, washed with 50 ml of Tris/Sorbitol buffer (40 mM TrisHCl pH=8.0; 1.2 M Sorbitol; 50 mM NaCl; 1 mM EDTA) and subjected to MNase digestion in 2 ml of NP-buffer (40 mM TrisHCl pH=8.0; 1.2 M Sorbitol; 50 mM NaCl; 1 mM EDTA, 0.1% NP-40, 0.75% Sigma Fungal Protease Inhibitor Cocktail; 10 mM _-mercaptoethanol; 5mM spermidine; 10 mM CaCl2) containing 150 u/ml of Nuclease S7 (Roche) for 5 min at 37¡ C. Reactions were stopped by addition of EDTA and EGTA mix to final concentration 25 mM each. Crosslinking was reversed overnight at 65¡ C in the presence of 0.5% SDS and 1 mg/ml proteinase K. DNA was purified by phenol/chloroform extraction and ethanol precipitation. RNA was removed by treatment with RNAseA/T1 mix (Fermentas). Purified DNA was resolved on 1% agarose gel and a band corresponding to mono-nucleosomal fragments was excised and subjected to gel-extraction.
DNA was end repaired with T4 DNA Polymerase (Invitrogen), T4 PNK (NEB) and DNA Pol I, Large Klenow fragment (Invitrogen), and an 'A' base was added using Klenow 3' to 5' exo minus (NEB). 1 pmol of barcoded adaptors were ligated on with T4 DNA ligase (Roche) and the products were PCR amplified with Phusion DNA Polymerase (NEB) and size selected by purification on 2% agarose-EX E-gels (Invitrogen) for fragments between 200-600 bp. Library size and concentration was determined by Bioanalyzer or Tapestation analysis (Agilent) and libraries were pooled equimolar amounts with up to 26 in each sequencing lane. Pooled libraries were gel purified twice, followed by column purification on MinElute columns (Qiagen). At least 10 nM were submitted for analysis on the Illumina Hi-Seq at Tufts University Core Facility.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
The sequenced reads were aligned using Bowtie allowing up to 10 matches ('-m 10 --best' options).
Paired end sequenced (PE) fragments with an inferred length between 90 bp and 200 bp were kept for downstream analysis.
A home-brew GC-content normalization, similar to BEADS, was applied to reduce variability in GC content of different samples. Instead of normalizing all samples to a flat GC distribution, the normalization brought the GC content of all samples similar to one of the samples, deemed to be midway. The final conclusions were independent of this normalization procedure.
The MNase-seq profile was calculated using a Gaussian Kernel smoothing (bandwidth=20 bp) on the positions of paired-end fragment centers.
Genome_build: ASM294v2
Supplementary_files_format_and_content: wig files show normalized smoothed fragment center density
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| Submission date |
Aug 06, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter J Park |
| E-mail(s) |
peter_park@harvard.edu
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| Phone |
617-432-7373
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| Organization name |
Harvard Medical School
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| Department |
Center for Biomedical Informatics
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| Street address |
10 Shattuck St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL13988 |
| Series (2) |
| GSE49572 |
Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast [Mnase-seq] |
| GSE49575 |
Spt6 regulates intragenic and antisense transcription, nucleosome positioning, and histone modifications genome-wide in fission yeast |
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| Relations |
| BioSample |
SAMN02303424 |
| SRA |
SRX331943 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1201969_MNase_WT_100Uml_N2_90_200bp_gcnorm19_KDE_bandw20sampled10.wig.gz |
2.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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