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| Status |
Public on Mar 11, 2023 |
| Title |
colB_mCIP_1 |
| Sample type |
genomic |
| |
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| Source name |
inflorescence buds
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
Strain;Wild type colmbia, Tissue; inflorescence buds, Age; 40days, Growth; on soil under continuous light condition (Enriched for methylated DNA at all sequenced contexts)
|
| Treatment protocol |
Untreated
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
genomic DNA was isolated from inflorescence buds using DNAeasy kit following instructions from the manufacturer (QIAGEN) instruction
|
| Label |
biotin
|
| Label protocol |
The genomic DNA in the samples was 3'-end-labelled with dd-UTP (Roche) by the terminal transferase (TdT, Roche) reaction for 1 h at 37 degrees C.
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| |
|
| Hybridization protocol |
Whole Genome Tiling Array was hybridized for 16 hrs, washed and stained with a streptavidin-phycoerythrin conjugate on the Affymetrix Fluidics Station 400 according to the standard Affymetrix protocol.
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| Scan protocol |
Whole Genome Tiling array were scanned on Affymetrix scanner G7
|
| Description |
Arabidopsis thaliana (ecotype Columbia) seeds were surface sterilized in 75% Ethanol 10 min, rinsed in distilled water. The seeds were cold-treated at 4 degrees C for 2 days and then incubated in the light for 40 days at 25 degrees C. An inflorescence buds were collected from mature Arabidopsis plants (40 days old) which were grown as described in the Arabidopsis Biological Resources Center manual (ABRC; http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm).DNA was extracted from the same tissues and at the same developmental stage as those used for methylome analyses using the Plant DNeasy Mini Kit (QIAGEN). The quality and quantity of isolated RNA and DNA was estimated by both gel electrophoresis and spectrophotometry.The methylcytosine immunoprecipitation (mCIP) method was described previously with slight modification (Zhang 2006). Immunoprecipitation was performed by incubating 5 ug of sonicated genomic DNA (approximately 300-500 bp length) with 10 ug mouse anti-methylcytosine monoclonal antibody (Calbiochem) using chromatin immunoprecipitation (ChIP) assay kit (Millipore) according to manufacturerÕs protocol. Then, eluted DNA was recovered by phenol-chloroform extraction and ethanol precipitation. The recovered DNA was examined real-time PCR to confirm methylated region of FWA promoter, AtSN1, and Ta3 and unmethylated region of actin8 in each fraction. The remaining DNA from the mock and elution fractions was amplified and used for microarray hybridization. Two biological replicates were performed for each genotype. The arrays were washed and stained using an Affymetrix fluidics station as instructed in the manufacturer's user manual. CELTM files from scanned arrays were used for further analysis by TileMap software.
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| Data processing |
Two biological replicates were performed for data processing each genotype and tissues with the method below, and yielding very consistent results (Box plot). Raw tiling microarray data processing was performed in a manner similar to previously described (Zhang et al., 2006). Methylated regions are expected to show a significantly higher hybridization signal in IP fraction when mock fraction is used as probe compared to IP fraction. First, the two groups (IP fraction and mock fraction) were subjected to quantile normalization. Next, we applied statistics method calculate the probe-level t-statistics using an empirical Bayes shrinkage estimator (Ji and Wong, 2005), to identify methylated regions and compared the results. Third, a two-state hidden Markov model (HMM) was trained to assign probes as either methylated or unmethylated. These three steps was performed using the software package TileMap with the HMM option (Ji and Wong, 2005). For the method, DNA methylated probes were called by applying a cutoff (posterior probability > 0.1 for TileMap) and DNA methylated regions were then derived by combining neighboring methylated probes allowing a maximal gap of 200 bases.(1). Regions statistically different between two groups were identified using the Hidden Markov Models (HMM) option. 1. H. Ji and W. H. Wong, Bioinformatics 21, 3629-3636 (2005).
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| |
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| Submission date |
Mar 03, 2008 |
| Last update date |
Mar 11, 2023 |
| Contact name |
Joseph Ecker |
| E-mail(s) |
josephecker@icloud.com
|
| Organization name |
The Salk Institute
|
| Department |
Genomic Analysis Laboratory
|
| Street address |
1001 North Torrey Pines Road
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
920009 |
| Country |
USA |
| |
|
| Platform ID |
GPL10978 |
| Series (1) |
| GSE10703 |
Genome-wide analyses of organ-specific DNA methylation and transcription in Arabidopsis thaliana |
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