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Status |
Public on Jul 15, 2009 |
Title |
Nucleosomes assembled on yeast and E. coli DNA by ACF |
Sample type |
SRA |
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Source name |
yeast and E. coli
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Organisms |
Escherichia coli; Saccharomyces cerevisiae |
Characteristics |
strain: Yeast BY4730 (MAT a, leu2∆0, met15∆0, ura3∆0) strain: E. coli JS5
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Extracted molecule |
genomic DNA |
Extraction protocol |
Yeast DNA was prepared from exponentially growing cells that were treated with Zymolyase. The resulting spherophasts were resuspended in Qiagen buffer G2 with 200μg/ml RNase A and then incubated with Proteinase K (8mg, Qiagen) for 30 minutes at 50C. After removal of cellular debris by centrifugation, DNA was precipitated with ethanol, isolated by spooling, and further purified using a Qiagen Genomic-tip 500/G according to the manufacturer’s instructions. E. coli DNA was isolated using a Qiagen Genomic-tip 500/G according to the manufacturer's instructions. Purified genomic DNA preparations from Saccharomyces cerevisiae as well as from Escherichia coli were sonicated separately to yield fragments that ranged in length from 5 to 10 kb. The resulting yeast and E. coli DNA samples were combined in a 3:1 mass ratio, and assembled into chromatin using a purified system containing recombinant Drosophila NAP-1 and ACF as well as purified native histones from Drosophila embryos. Chromatin was extensively digested with micrococcal nuclease to yield core particles. The ~147 bp DNA fragments derived from the core particles were purified by agarose gel electrophoresis. Mononucleosomal DNA was treated with calf intestinal alkaline phosphatase (New England Biolabs) to remove 3’ phosphate groups left after the micrococcal nuclease treatment. Libraries were prepared according to Illumina’s instructions accompanying the DNA Sample Kit (Part# 0801-0303) and sequenced on the Genome Analyzer following the manufacturer’s protocols.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer |
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Description |
n/a
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Data processing |
Sequence tags were obtained and aligned to the Saccharomyces cerevisiae from SGD2 (Apr 2008 build) and Escherichia coli K12 MG1655 (U00096) genomes using the Illumina/Solexa Analysis Pipeline. The analysis allowed 2 mismatches per mapped read and only uniquely aligned reads were retained. In sample 1 (Nucleosomes assembled on yeast and E. coli DNA by salt dialysis) and sample 2 (Nucleosomes assembled on yeast and E. coli DNA by ACF), each sequencing tag represents one end of a mono-nucleosomal DNA fragment. Therefore, we extended each tag to 146 bp in the 3’ direction to represent a whole mono-nucleosome, and then we piled up all extended sequencing tags in a sample to obtain the nucleosome density profile along the genome. The identical approach was also used in sample 3 (Yeast-Ecoli control, sonicated) to serve as control.
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Submission date |
Mar 11, 2009 |
Last update date |
May 15, 2019 |
Contact name |
Ghia Euskirchen |
E-mail(s) |
ghia.euskirchen@stanford.edu
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Organization name |
Stanford University
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Department |
Genetics
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Lab |
Snyder
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Street address |
1501 S. California Ave.
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City |
Palo Alto |
State/province |
CA |
ZIP/Postal code |
94304 |
Country |
USA |
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Platform ID |
GPL10839 |
Series (1) |
GSE15188 |
Intrinsic histone-DNA interactions are not the major determinant of nucleosome positions in vivo |
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Relations |
SRA |
SRX012148 |
BioSample |
SAMN00004271 |
Supplementary file |
Size |
Download |
File type/resource |
GSM379240_ACF_Ecoli_146bp.wig.gz |
1.5 Mb |
(ftp)(http) |
WIG |
GSM379240_ACF_eland_result_laneA_FC12187_102707_s_3.txt.gz |
149.6 Mb |
(ftp)(http) |
TXT |
GSM379240_ACF_eland_result_laneB_FC200RR_20080220_s_1.txt.gz |
199.9 Mb |
(ftp)(http) |
TXT |
GSM379240_ACF_yeast_146bp.wig.gz |
4.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
Processed data provided as supplementary file |
Raw data are available in SRA |
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