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| Status |
Public on Sep 26, 2012 |
| Title |
H3shutoff-MNase-3hr-rep1 |
| Sample type |
SRA |
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| Source name |
H3shutoff-MNase-3hr
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: DCB200.1
carbon source: dextrose
time point: 3 hours
od: 0.8-1
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| Treatment protocol |
Cells were collected by filtration using a 0.2um filter, washed with media containing 1% yeast extract, 2% peptone, 2% dextrose, and resuspended at the intial concentration in fresh media containing 1% yeast extract, 2% peptone, 2% dextrose.
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| Growth protocol |
Cells were grown with shaking at 30C in media containing 1% yeast extract, 2% peptone, 2% galactose and grown to an OD600 of between 0.8 and 1.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde for 15 minutes at room temperature with shaking. Crosslinking was quenched by addition of glycine to a final concentration of 0.125 M and incubation at room temperature for 5 minutes. Cells were then spun down at 3000 rpm in a Sorvall RT-34 centrifuge, washed twice with 20 mL of 1 M sorbitol, and resuspended in a final volume of 10 mL in spheroplast digestion buffer [1 M sorbitol, 1mM 2-mercaptoethanol (Fischer Scientific BP176100) and 42000 U of lyticase (Sigma-Aldrich L2524)]. Cells were rocked at 30C for 15 minutes, spun down at 3000 rpm in a Sorvall RT-34 centrifuge, and washed twice with 10 mL of 1 M sorbitol. Spheroplasts were resuspended at 0.12 g spheroplast/mL of MNase digestion buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris pH 7.5, 5 mM MgCl2, 1 mM CaCl2, 0.075% NP-40 with 1 mM 2-mercaptoethanol and 0.5 mM spermidine added immediately before use). 600ul aliquots were digested witha titration of micrococcal nuclease A (Worthington-Biochemical LS004797) ranging from 0 to 50U for 15 minutes at 37C. Digestion was stopped with 150ul stop buffer (5% SDS, 50 mM EDTA). 5ul of a 20 mg/mL stock solution of Proteinase K (Roche 03 115 879 001) was added and the tube incubated at 65C overnight to reverse crosslinking. DNA was isolated by standard phenol:chloroform extraction (2x) and precipitated using isopropanol. DNA was dried and resuspended in 15ul of ddH2O. 1ul of previously boiled 10 mg/mL RNase A (Sigma Aldrich R5503) was added and the reaction incubated at 37C for 30-60 minutes to digest RNA. The DNA was then loaded on a 2% agarose gel, run out, and the band corresponding to a mono-nucleosome band in the sample showing clear mono-, di- and tri-nucleosome bands gel extracted using a dark reader (Clare ChemicalResearch DR46B Transilluminator). Samples were prepared using a modified version of the Illumina Solexa preparation protocol. DNA was blunted using the END-IT DNA Repair Kit (Epicentre ER0720). 50 ng of mononucleosome DNA was incubated with 5ul 10x buffer, 5ul dNTP mix, 5ul ATP solution and 1ul enzyme mix in a 50ul reaction for 60 minutes at room temperature. DNA was recovered using the QIAQuick Gel Extraction kit (Qiagen 28704) with 50ul ddH2O and 400ul of QG buffer. DNA was eluted using 2 15ul elutions and then adenylated using 1ul of NEB exo- klenow (M212M), 5ul Klenow buffer and 1ul 10mM dATP in a 50ul reaction for 60 minutes at room temperature. DNA was isolated using a QIAquick MinElute kit (Qiagen 28006) and eluted in 10ul of EB. Adapters were ligated using NEB Quick T4 DNA Ligase (M2200S) with a 10:1 dilution of adapters provided by the UNC High Throughput Sequencing Facility (UNC HTSF) in a 50ul reaction containing 10ul of DNA, 25ul 2X Quick Ligation Buffer, 2uL adapters and 1ul DNA ligas for 25 minutes at room temperature. DNA was purified using a QIAQuick MinElute column and eluted in 10ul of EB. DNA was amplified using Stratagene PfuUltra II Fusion HS DNA Polymerase (Stratagene 600670) using 2ul DNA, 2ul Solexa single-end PCR primers, 10ul 10X PfuUltra II buffer, 10ul 2.5 mM dNTP and 1ul of Pfu Ultra II DNA polymerase in a 100uL reaction with the following cycling conditions: 95C for 1 minute; 18 cycles of 95C for 50 seconds, 65C for 60 seconds, 72C for 30 seconds; 72C for 5 minutes; 4C hold. DNA was purified using the QIAQuick MinElute kit in 10ul of buffer EB and run out on a 2% agarose gel. The band corresponding to mononucleosome + adapters (approximately 250 base pairs) was cut out on the dark reader, gel extracted using the QIAQuick Gel Extraction kit, and submitted to UNC HTSF for sequencing.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
MNase digestion of H3 shutoff cells after H3 depletion following 3 hours in dextrose
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| Data processing |
WIG files - Reads were aligned to the sacCer1 version of the yeast genome using Bowtie v. 0.12.6 with default settings. Single end reads were extended to the average fragment size of the experiment. Reads aligning to the rDNA locus (chr12 450,000-472,5000) were removed and 5 million reads were randomly selected from the remaining, aligned reads to generate the final, selected reads for each experiment.
TXT files - A dyad density map was created for each experiment using the center of each mapped, selected read. The dyad density map was Gaussian smoothed with a s.d. of 10 bp and a window of 3 s.d. For each chromosome, the smoothed dyad density maximum was selected as the center of a nucleosome. The protected region for the nucleosome was calculated by averaging the fragment length of all reads covering the called nucleosomeâs dyad. The s.d. of the dyad was calculated using the raw dyad counts that fell within the nucleosomeâs protected region. All bases within 1 protected region of the nucleosome dyad were set to a smoothed dyad density of 0 and the process repeated until no remaining nucleosomes could be called. Nucleosome occupancy was defined as the number of read centers falling within a 100 bp window centered on the nucleosome dyad.
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| Submission date |
May 14, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jason D Lieb |
| E-mail(s) |
[email protected]
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| Phone |
919-843-3228
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| URL |
http://www.bio.unc.edu/faculty/lieb/labpages/default.shtml
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| Organization name |
University of North Carolina at Chapel Hill
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| Department |
Biology
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| Lab |
Jason Lieb
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| Street address |
203 Fordham Hall
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
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| Platform ID |
GPL13272 |
| Series (2) |
| GSE29291 |
Effects of Histone H3 depletion on nucleosome occupancy and positioning through the S. cerevisiae genome [single-end MNase-seq] |
| GSE29294 |
Effects of Histone H3 depletion on nucleosome occupancy and positioning through the S. cerevisiae genome |
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| Relations |
| SRA |
SRX063262 |
| BioSample |
SAMN00618977 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM724100_AG204_5milFMR_nucCalls.txt.gz |
1.6 Mb |
(ftp)(http) |
TXT |
| GSM724100_AG204_SE_5milFMR.bed.gz |
15.3 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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