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| Status |
Public on Dec 07, 2011 |
| Title |
OverproducingH2A_15min_(20101103_B5_KK_H2A) |
| Sample type |
SRA |
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| Source name |
DNA from H2A-FLAG chromatin immunoprecipitation
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: Sby8796
medium: YEPD grown:OD=0.8
antibody: Sigma A2220 Anti Flag M2 Affinity gel; lot 020M6014
sample type: MNase-protected DNA fragments from H2A-FLAG immunoprecipitation of native chromatin, 15 minute digestion
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Yeast nuclei were prepared as described [Furuyama, S., and Biggins, S. (2007), Centromere identity is specified by a single centromeric nucleosome in budding yeast, Proc Natl Acad Sci USA 104, 14706-14711], flash-frozen in liquid nitrogen and stored at -80oC. Nuclei were thawed at room temperature and digested with MNase followed by chromatin preparation as described [Furuyama and Biggins, (2007)], except that after MNase digestion, the slurry was passed four times through a 20 guage needle, then four times through a 26 guage needle [Jin, C., and Felsenfeld, G. (2007), Nucleosome stability mediated by histone variants H3.3 and H2A.Z, Genes Dev 21, 1519-1529], and the combined S1+S2 supernatants were clarified by centrifugation in a fixed-angle Sorvall SS34 rotor at 12,000 rpm at 4oC for 10 min at least twice or until no visible pellet remained. ChIP was performed as previously described [Furuyama and Biggins, (2007)], and DNA was extracted using a standard protocol [Mito, Y., Henikoff, J., and Henikoff, S. (2005), Genome-scale profiling of histone H3.3 replacement patterns, Nat Genet 37, 1090-1097]. Standard Illumina protocol wa used Catalog #PE-930-1001, except size selected 100bp to 300bp.
For Flag IP, we used Sigma A2220 Anti Flag M2 Affinity gel; lot 020M6014.
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
MNase-protected DNA fragments from H2A-FLAG immunoprecipitation of native chromatin, 15 minute digestion
Illumina HiSeq 2000
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| Data processing |
1. We used Novoalign to map paired-end reads to release 61 (UCSC sacCer2) of the S.cervisiae genomic sequence obtained from downloads.yeastgenome.org. 50bp reads were trimmed to 25bp. If a read was mapped to multiple locations, one location was picked at random. (Supplementary file KK_H2A.Novoalign.sam) 2. For each base pair in the genome, we counted the number of paired-end fragments aligned over it. 3. We normalized base pair counts by dividing by the total number of counts for all base pairs and then multiplying by the total number of base pairs in the genome. (Supplementary file KK_H2A.wig) 4. We broke down aligned paired-end fragments into sub-groups by insert size length and repeated steps 2. and 3. for the paired-end fragments in each sub-group.
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| Submission date |
Apr 06, 2011 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Jorja Henikoff |
| E-mail(s) |
[email protected]
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| Phone |
206-667-4850
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| Organization name |
Fred Hutchinson Cancer Research Center
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| Department |
Basic Sciences
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| Lab |
Henikoff
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| Street address |
1100 Fairview AV N, A1-162
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109-1024 |
| Country |
USA |
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| Platform ID |
GPL13821 |
| Series (1) |
| GSE28298 |
Tripartite organization of centromeric chromatin in budding yeast |
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| Relations |
| BioSample |
SAMN02198543 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM702298.wig.gz |
68.6 Mb |
(ftp)(http) |
WIG |
| GSM702298_henikoff_KK_H2A.Novoalign.sam.gz |
2.4 Gb |
(ftp)(http) |
SAM |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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