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| Status |
Public on Dec 18, 2008 |
| Title |
In_vitro (2 biological replicates) |
| Sample type |
SRA |
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| Source name |
S. cerevisiae genomic DNA and histone octamer from chicken erythrocytes
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
yeast strain: YLC8 [MAT(a) ura3(∆) leu2(∆) his3(∆) met15(∆)]
genomic dna: purified DNA
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| Treatment protocol |
As indicated in sample protocols
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| Growth protocol |
S. cerevisiae genomic DNA was purified from strain YLC8 using standard methods with additional methods to remove contaminating RNA. After recovery by ethanol precipitation, the sample was resuspended in TE buffer and the DNA concentration determined by agarose gel electrophoresis using ethidium stain followed by comparison to mass standards using quantitative fluorometry. The sample was then digested with 100µg of RNase A for every 10µg of DNA overnight at 50C and then the DNA was ethanol precipitated. After resuspension in TE the genomic DNA was sheared twice each through a 25 gauge and then 27.5 gauge needle. The mixture was then run out on a 1% agarose gel at 100V for 6-8 hours. The genomic DNA band was cut out and the agarose slab was re-electrophoresed inside a dialysis bag to elute DNA. The resulting DNA was then reconstituted into nucleosomes by salt gradient dialysis using 40µg histone octomer + 100 µg DNA in 200uL volume. The resulting nucleosomes were then isolated by micrococcal nuclease digestion using 6x10^-3 units MNase per 10µg reconstituted DNA in 10mM Tris pH 8.0, 1mM CaCl2 for 5 min at 37C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Samples were extracted as indicated in sample protocols. All samples were then blunted using END-IT DNA Repair Kit #ER0720 from Epicentre (< 5µg DNA, 5uL 10x buffer, 5uL dNTP mix, 5uL ATP solution, 1uL enzyme mix, water to 50uL. Incubated at room temp for 60 minutes, then cleaned up using Qiagen buffer QG on QiaQuick columns with 2 PE buffer washes, eluted with 30uL EB), then A-tailed using #KL06041K from Epicentre (Klenow Exo-minus) (30uL DNA solution, 5uL Klenow, 1uL 10mM dATP, 1uL Klenow, water to 50uL, 60 min at room temp, then cleaned up with Qiagen MinElute columns and eluted in 10uL EB). Solexa adapters were ligated using a Fast Link Kit (#LK11025 from Epicentre) (10uL DNA, 1.5uL 10x buffer, 0.75 uL 10mM ATP, 1uL of Solexa adapters, 1uL ligase, and water to 50uL incubated for 60 min at room temp, then 7.5uL water, 1uL 10x buffer, 0.5uL 10mM ATP, and 1uL ligase were added and the entire reaction incubated at 16C overnight before purification on QiaQuick columns as in blunting reaction). Samples were run out on a 2% agarose gel and the mononucleosome sized band (~147bp) was extracted using Qiagen Gel Extraction columns. Samples were amplified by PCR (Stratagen PfuUltra II Fusion #600670) (30uL ligated DNA, 2uL Solexa PCR primers, 10uL 10x buffer, 10uL 2.5mM dNTP, 1uL enzyme, water to 100uL for PCR [95C 1 min, (95C 50'', 65C 1', 72C 30'') x 15 cycles, 72C 5min, 4C hold] and then cleaned on QiaQuick columns) before being sequenced on Illumina Genome Analyzer II machines.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
library source : genomic
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| Data processing |
Mapped Reads: Unique reads mapped to the yeast genome, and extended to the average fragment length in the experiment (140-170 bp).
Normalized Tracks: For each Sample, we used the map reads to calculate at every basepair the log-ratio between the number of reads that cover that basepair and the average number of reads per basepair across the genome. We then set the genomic mean in each sample to zero by subtracting the genome-wide mean from every basepair. Finally, we averaged the replicates within each experimental condition, resulting in four tracks of log-normalized nucleosome coccupancy. One file for each biological condition, four files in total.
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| Submission date |
Dec 16, 2008 |
| Last update date |
May 15, 2019 |
| Contact name |
Desiree Tillo |
| E-mail(s) |
desiree.tillo@nih.gov
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| Phone |
+1-240-760-7289
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| Organization name |
NIH/NCI
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| Street address |
41 Center Dr, Room D310
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL9377 |
| Series (1) |
| GSE13622 |
The DNA-Encoded Nucleosome Organization of a Eukaryotic Genome |
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| Relations |
| SRA |
SRX008314 |
| BioSample |
SAMN02195536 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM351491_InVitro_MS_R1.tab.gz |
13.2 Mb |
(ftp)(http) |
TAB |
| GSM351491_InVitro_R2.tab.gz |
13.8 Mb |
(ftp)(http) |
TAB |
| GSM351491_InVitro_normalized.tab.gz |
37.4 Mb |
(ftp)(http) |
TAB |
| GSM351491_SRA_README.txt.gz |
91 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
| Processed data included within Sample table |
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