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| Status |
Public on May 29, 2010 |
| Title |
Arabidopsis Mnase-Seq (seq, prb) |
| Sample type |
SRA |
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| Source name |
Arabidopsis shoot tissue
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| Organism |
Arabidopsis thaliana |
| Characteristics |
strain: Columbia-0
genotype: wild type
tissue: shoots
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| Treatment protocol |
Arabidopsis shoot tissues were obtained.
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| Growth protocol |
For MNase and ChIP-Seq samples, Arabidopsis plants were grown under continuous light for three weeks.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For MNase samples, Arabidopsis nuclei were prepared as described previously (Bernatavichute et al. 2008 PLoS ONE 3: e3156) with the following modifications. After spinning the nuclei pellet at 3000 rpm for 5 min (SS-34, Sorvall), the pellet was resuspended in 5 ml of HBB buffer (25 mM Tris-Cl pH 7.6, 0.44 M sucrose, 10 mM MgCl2, 0.1% Triton-X, 10 mM beta-mercaptoethanol), applied to a 40/60% Percoll (GE Healthcare) gradient in HBB and spun 2000 rpm for 20 min (SS-34, Sorvall). Isolated nuclei were washed two times in HBB buffer and flash frozen in liquid nitrogen in HBC buffer (25 mM Tris-Cl pH 7.6, 25 mM Tris-Cl pH 7.6, 440 mM sucrose, 10 mM MgCl2 and 0.1% Triton-X, 10 mM beta-mercaptoethanol, 20% glycerol). Nuclei from 1/5 of each preparation were treated with four ul of RNAse A, 10 ug/ul, (Qiagen) and digested with 0.02 U/ul (final concentration) of Micrococcal Nuclease (Takara), 3 min. Digestion was done in digestion buffer (16 mM Tris-Cl, pH 7.6, 50 mM NaCl, 2.5 mM CaCl2, 0.01 mM PMSF and EDTA-free protease inhibitor cocktail (Roche)) and stopped with 10 mM EDTA and treated with Proteinase K (Roche). DNA was purified either with phenol:chlorophorm extraction and precipitated with salts and ethanol or with ChIP DNA Clean and Concentrator kit (Zymo Research). Purified DNA was run on 2% agarose gel and bands corresponding to ~150 bp were cut and purified with a Gel Purification kit (Qiagen). Approximately 300ng of MNase digested mononucleosome DNA were used for library generation following manufacturer protocols (Illumina). Libraries were sequenced according to manufacturer instructions (Illumina). For H3-ChIPSeq samples, chromatin was immunoprecipitated using an antibody against histone H3 as previously described (Bernatavichute et al. 2008 PLoS ONE 3: e3156). Libraries were constructed and DNA was sequenced according to manufacturer instructions (Illumina). For BS-seq samples, the HSF1 hESC line was cultured as described previously (Xiao et al. 1999 Embo J 18: 5943-5952). BS-Seq DNA library construction, high-throughput Illumina sequencing by and analysis were performed similar to that previously described (Cokus et al. 2008 Nature 452: 215-219).
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| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina Genome Analyzer |
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| Description |
MNase digested DNA was size selected for the mononucleosome fraction
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| Data processing |
For MNase samples, Bowtie was used to align the reads to the TAIR7 genome. Alignments with greater than 3 mismatches were filtered and only unique alignments were included. The processed file indicates the position, strand, and number of reads alingning to that position. For the ChIPSeq sample, reads were mapped to the TAIR7 genome using a probabilistic algorithm previously described (Cokus et al. 2008 Nature 452: 215-219) . The processed file indicates the position, strand, and number of reads alingning to that position. BS-seq was analyzed similarly as previously described (Cokus et al. 2008 Nature 452: 215-219). The processed file indicates the chromosome, strand, position, fraction methylated, and total number of counts (methylated Cs and unmethylated Cs). All processed files indicate one-based positions (i.e. first base of the chromosome corresponds to position 1).
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| Submission date |
May 13, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
matteo Pellegrini |
| E-mail(s) |
matteop@mcdb.ucla.edu
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| Phone |
310-825-0012
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| Organization name |
UCLA
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| Department |
MCD Biology
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| Lab |
Matteo Pellegrini
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| Street address |
621 Charles Young Drive South
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
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| Platform ID |
GPL9062 |
| Series (2) |
| GSE21673 |
Relationship between nucleosome positioning and DNA methylation |
| GSE21821 |
Relationship between nucleosome positioning and DNA methylation: Bisulfite-Seq, ChIP-Seq, and Mnase-Seq |
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| Relations |
| SRA |
SRX021423 |
| BioSample |
SAMN00014124 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM543295_ArabidopsisMNase1.txt.gz |
71.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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