|
| Status |
Public on Nov 30, 2007 |
| Title |
BiMP met1-3 BS+ |
| Sample type |
genomic |
| |
|
| Source name |
BiMP met1-3 BS+
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Genotype: M/M; treatment: bisulfite +
|
| Treatment protocol |
CTAB extracted DNA samples were Rnase A and Proteinase K treated (Warnecke et al. 2002), phenol extracted (Sigma) and then ethanol precipitated. DNA samples (4.0μg) were digested with a 2-fold excess of DraI restriction enzyme (Promega), as recommended by the manufacturer, and phenol extracted (Sigma) and ethanol precipitated.
|
| Growth protocol |
Plants were grown in soil under under 8 hr light/16 hr dark. Leaf tissue from 9-12 individuals was pooled per DNA sample. Thre replicate samples per entry were collected.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For the bisulfite treatment, the EpiTect Bisulfite modification kit (Qiagen) was used as directed by the manufacturer.
|
| Label |
biotin
|
| Label protocol |
DNA was amplified using our BiMP amplification method. DNA labeling reaction with terminal deoxynucleotidyl transferase and DNA labeling reagent (Affymetrix) were added to each DNA sample
|
| |
|
| Hybridization protocol |
Following a 2 hour fragmentation, 9 ug of DNA were hybridized for 16 hr at 45C on Arabidopsis Tiling 1.0R arrays . GeneChips were washed and stained as directed by Affymetrix
|
| Scan protocol |
GeneChips were scanned using the GeneChip® Scanner 3000 7G
|
| Description |
one sample analysis (met_bs+1, met_bs+2, met_bs+3)
|
| Data processing |
Arabidopsis data (CEL) files were analyzed using the Tiling Analysis Software (TAS, Affymetrix) using the following parameters. The normalization parameters were scaled to an intensity of 100 on a log2 scale and the p-value was -10log10. The probe analysis was set to a bandwidth of 80 and perfect match/mismatch intensities were analyzed using a one-sided upper test. The interval analysis on intensity values was set to a threshold of 1-e5 with a maximum gap of 80 and a minimum run to 40, resulting in a “sliding window” analysis at a 161 bp length. For analyzing methylation polymorphisms, hypo- and hypermethylated intervals were calculated using TAS. Per entry, the three biological replicate bisulfite-treated DNA files and the three biological replicate non treated DNA files were selected as the treatment and control group files, respectively, in a two sample comparison analysis using quantile normalization of both groups together. The resulting ColBS+ and met1-3BS+ BAR files, for the above two-sample comparisons, were loaded into IGB (Affymetrix). Since IGB performs only a one-sided statistical test, hypomethylated and hypermethylated comparisons were made separately. The hypomethylation graph was the difference in the log2 scaled (Col_BS+) from (met1-3_BS+) results and the hypermethylation was the difference in the log2 scaled (met1-3_BS+) from (Col_BS+). The graphs of these comparisons were adjusted to zero and the positive cutoff of 4.0 was applied. Additionally, the three CEL files per entry were analyzed in TAS using the one sample detection analysis to create a single BAR files for each entry alone. The one sample parameter were as above and allowed further visual inspection and comparison.
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| |
|
| Submission date |
Sep 14, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Jon Reinders |
| E-mail(s) |
jon.reinders@bioveg.unige.ch
|
| Phone |
+41 22 379 3029
|
| Fax |
+41 22 379 3107
|
| URL |
http://www.unige.ch/sciences/biologie/plantsciences/grpaszkowski/
|
| Organization name |
Universite de Geneve
|
| Department |
Department of Plant Biology
|
| Lab |
Laboratory of Plant Genetics
|
| Street address |
30 quai Ernest-Ansermet
|
| City |
Genève |
| State/province |
Genève |
| ZIP/Postal code |
CH-1211 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL10977 |
| Series (2) |
| GSE9051 |
Genome-wide, High Resolution DNA Methylation Profiling Using Bisulfite-Mediated Cytosine Conversion |
| GSE13438 |
Bisulfite Methylation Profiling (BiMP) analysis of met1-3 epigenetic Recombinant Inbred Lines (epiRILs) |
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