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| Status |
Public on Nov 12, 2013 |
| Title |
Spombe_Exp2 paired-end |
| Sample type |
SRA |
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| Source name |
Nucleosomal DNA
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain: H4S48C: hhf1S48C, hhf2S48C
growth phase: Mid log phase growth
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| Treatment protocol |
For a 500mL culture, cells were collected and quickly washed twice with water followed by a 10 minute incubation at 30 degrees Celcius in pre-incubation buffer (20 mM citric acid, 20 mM Na2HPO4, 30 mM β- mercaptoethanol (BME), 40 mM EDTA). Cells were centrifuged and resuspended in permeabilizing buffer (1M sorbitol, 50 mM Tris/HCl pH7.4, 5mM BME with 5mg of lyticase (Sigma, L5263vc-200KU) using 10 ml per gram of original cell pellet and spheroplasted at 30 °C with gentle shaking. Spheroplast formation was terminated at an OD600 of 30–40% of the original value by centrifugation at 4°C. Spheroplasts were washed twice in 5mL of 1M sorbitol, 50 mM Tris/HCl and resuspended in 2mL of labeling buffer (1M sorbitol, 50mM NaCl, 10mM Tris-HCl pH 7.5, 5mM MgCl2, 0.5mM spermidine, 0.15mM spermine, 0.075% NP-40). The thiol reactive label - N(1,10 phenanthroline- 5-yl) iodoacetamide (Biotium cat# 92015), was dissolved in DMSO to a concentration of 7mM and added to the cells for a final concentration of 1.4mM of label and 20% total volume of DMSO. The light-sensitive labeling reaction was incubated at room temperature, with rotation for 2 hours. This reaction was then washed 2x in 10mL of 1M sorbitol and 0.1% NP-40, then resuspended in 1.5mL mapping buffer (1M sorbitol, 2.5mM NaCl, 50mM Tris-HCl pH 7.5, 5mM MgCl2, 0.5mM spermidine, 0.15mM spermine and 0.1% NP-40). Cupric chloride (CuCl2) was added to a final concentration of 150mM and incubated for 5min. The cells were washed 2x in 10mL of mapping buffer and resuspended in 1.5mL mapping buffer. Beta-mercaptopropionic acid (MPA) was added to a final concentration of 6mM, and incubated for 5min, reducing the copper to cuprous chloride. The chemical mapping reaction was then initiated by the addition of hydrogen peroxide to a final concentration of 6mM. The reaction was mixed well and incubated for 20 minutes. The mapping reaction was quenched by the addition of Neocuprine (Sigma), 2.8mM final. Once quenched the cells were spun down and resuspended in 1mL of 1mM Tris-HCl pH 7.5 and 0.1mM EDTA. To disrupt histone-DNA contacts, SDS and NaCl were added to a final concentration of 2% and 2M respectively and incubated for 1 hour.
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| Growth protocol |
The H4S48C S. pombe strain was grown at 30 degrees Celcius in YES medium (5 g/L yeast extract, 30 g/L glucose and 225 mg/L adenine, histidine, leucine, racil and lysine hydrochloride) to mid log phase (OD600 0.7).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was purified by phenol-chloroform extraction and subsequent ethanol precipitation. The sample was RNased (1mg/ml final) overnight at 42oC and run on a 2% agarose gel. The shortest DNA band, corresponding to the center-to-center distance of two nucleosome neighbors, was cut out of gel and the DNA was extracted. The ends of the chemically mapped DNA were blunted using first NEB’s Klenow Polymerase large fragment (cat # M0210L) then processed further using Lucigen’s DNATerminator End Repair Kit (cat # 40035-2). ABI SOLiD adapters were ligated to the blunted and phosphorylated DNA ends. Nick translation and PCR amplification were completed following ABI’s SOLiD Fragment Preparation protocol. All samples were sequenced with ABI’s SOLiD next generation sequencing system.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
AB SOLiD 5500 |
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| Description |
KGY425 (ATCC 96155) (h- his3-D1 leu1-32 ura4-D18 ade6-M210)
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| Data processing |
barcodes were removed first.
Paired-end reads were aligned by allowing for 0 to 3 mismatches progressively. If any read was aligned for the given mismatch tolerance, it was not re-aligned in the following steps.
For unaligned reads, we trimmed the last four bases, and repeated last step.
For unaligned reads, we aligned each end (untrimmed) separately by allowing 0 to 2 mismatches progressively.
For unaligned reads, we further trimmed the last four bases, and repeated the single-end alignment by allowing up to 1 mismatch
The alignment results from above steps were combined.
Genome_build: PomBase version 16.28, released on 03-10-2012
Supplementary_files_format_and_content: tab delimited text file
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| Submission date |
May 15, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ji-Ping Wang |
| E-mail(s) |
jzwang@northwestern.edu
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| Organization name |
Northwestern University
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| Street address |
2006 Sheridan Road
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| City |
Evanston |
| ZIP/Postal code |
60208 |
| Country |
USA |
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| Platform ID |
GPL17205 |
| Series (1) |
| GSE46975 |
Chemical map of S. pombe reveals species-specific features in nucleosome positioning |
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| Relations |
| BioSample |
SAMN02179316 |
| SRA |
SRX286745 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1142338_Spombe_Exp2_alignment.txt.gz |
944.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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