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| Status |
Public on Jul 11, 2013 |
| Title |
Total mononucleosomes |
| Sample type |
SRA |
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| Source name |
Yeast cells, total mononucleosomes
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: yFR212
background: W303
genotype/variation: MATa, ade2-1, trp1-1, can1-100, leu2-3,112, his3-11,15, ura3-1, GAL+, psi+, HTZ1::3myc
chip antibody: none
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| Growth protocol |
Two hundred mL of yeast cells were grown in YPD to an OD600 of 0.8, crosslinked for 15 minutes with 1% formaldehyde and quenched for 5 minutes with 125mM glycine. The culture was split into four 50mL Falcon tubes, centrifuged for 5 minutes at 3,000rpm, resuspended in H2O and pooled into two Falcon tubes. Cells were centrifuged again and each dry yeast pellets was individually snap frozen in liquid nitrogen and stored at -80˚C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were centrifuged again and the dry yeast pellets were snap frozen in liquid nitrogen and stored at -80C. One cell pellet was kept as a backup and the other one was thawed and resuspended in 20 mL of buffer Z (1M Sorbitol, 50mM Tris pH 7.4) before adding 14 μL of β-mercaptoEtOH and 0.5 mL of zymolase solution (1mg/mL, freshly prepared in buffer Z). Cells were shaken for 40 minutes at 30˚C and centrifuged 10 minutes at 1,500 rpm. The supernatant was aspired and discarded while the pellet was resuspended in 1.25 mL of NP buffer (50mM NaCl, 10mM Tris pH7.4, 5mM MgCl2, 1mM CaCl2, 0.075% NP40, 0.5mM spermidine, 1mM β-mercaptoEtOH). After this one, one of two different protocols was applied.
Three hundred μL aliquots (five) were treated with different amounts of MNase for 20 minutes at 37 C and reactions were stop by adding 6μL of 500mM EDTA.
For total nucleosomes, one 150 μL from each reaction was treated with 38 μL of 5xTE/SDS (50mM Tris pH8, 5mM EDTA, 5% SDS) overnight at 65˚C to reverse the crosslink. The remaining of the extract was kept as a backup at -20˚C. The next day, 210 μL of RNase mix (205 μL TE, 3 μL RNaseA, 2 μL glycogen) was added, followed by an incubation of two hours at 37˚C. 8 μL of proteinase K (from 20mg/mL stock) was added and incubation was pursued for an additional two hours at 37˚C. DNA was purified by phenol:chloroform extraction, EtOH precipitated and resuspended in 50 μL of 10mM Tris pH 8.0. Inspection on agarose gel revealed some residual RNA so an additional RNaseA treatment, followed by phenol:chloroform extraction and EtOH precipitation was performed. Aliquots were inspected on 2% agarose gel and the sample contains mainly monononucleosomes.
The library was prepared by following the paired-end library protocol (Illumina Inc., USA). Briefly, the DNA was subject to end-repair, and phosphorylation by T4 DNA polymerase, Klenow DNA Polymerase, and T4 polynucleotide kinase respectively in a single reaction, and then 3' A overhangs generation by Klenow fragment (3' to 5' exo minus), and ligated to Illumina PE adapters, which contain 5' T overhangs. The adapter-ligated products were purified on QIAquick spin columns (Qiagen), then PCR-amplified with Phusion DNA Polymerase in 10-15 cycles using Illumina's PE primer set (Illumina). PCR product of desired size range was purified using a 8% PAGE, and the DNA quality was assessed and quantified using an Agilent DNA 1000 series II assay and Nanodrop 7500 spectrophotometer (Nanodrop, USA) and diluted to 10nM. The final concentration was double checked and determined by Quant-iT dsDNA HS Assay Kit using Qubit fluorometer (Invitrogen). Clusters were generated on the Illumina cluster station and sequence was run on an Illumina Genome Analyzer IIx following the manufacturer's instructions.
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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 |
SC0016_61YDGAAXX_7_TCATTC
Strain yFR212 is in a W303 background where the HTZ1 gene is tagged with 3 Myc epitopes in its 3' end (C-terminal of the Htz1 protein).
MNase-Seq
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| Data processing |
BWA was used for alignment.
samtools was used for converting fastq format to BAM format.
Data were called and filtered using R (development version).
R/Bioconductor package PING version 2.0.0 was used for peak-calling. We did run PING for PE and SE options. The option is shown in the Processed data file names : "_PE/SE_".
Genome_build: sacCer2
Supplementary_files_format_and_content: Tab-delimited text files. The processed data files are outputs of the PING software, which was developed to identify nucleosomes positions. The output includes the information for each identified nucleosome: chromosome, the coordinate of center of the nucleosome, estimated parameter (indication of uncertainty), enrichment score, range of the identified nucleosome region, and rank based on the enrichment score.
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| Submission date |
May 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Sangsoon Woo |
| E-mail(s) |
swoo@fhcrc.org
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| Organization name |
Fred Hutchinson Cancer Research Center
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| Street address |
1100 Fairview Ave N
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL13272 |
| Series (1) |
| GSE47073 |
PING 2.0: An R/Bioconductor package for nucleosome positioning using next-generation sequencing data |
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| Relations |
| BioSample |
SAMN02146698 |
| SRA |
SRX282124 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1144378_PING_SC0016_61YDGAAXX_7_1_chrIII_ONLY_PE.txt.gz |
232.7 Kb |
(ftp)(http) |
TXT |
| GSM1144378_PING_SC0016_61YDGAAXX_7_1_chrIII_ONLY_SE.txt.gz |
186.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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