|
| Status |
Public on Feb 18, 2013 |
| Title |
T4 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
mCIP DNA from A. thaliana crown galls
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: crown galls from Agrobacterium tumefaciens strain C58noc inoculated inflorescence stalks
antibody: 5-methylcytosine mouse monoclonal (Diagenode, cat# MAb-006-100, lot# DA-0018)
|
| Growth protocol |
Arabidopsis plants were cultivated in growth chambers at 22°C during the light and 16°C during the dark period in 12 h intervals. Arabidopsis plants were cultivated in growth chambers at 22°C during the light and 16°C during the dark period in 12 h intervals.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tissue was separated from the host inflorescence stalk under a stereozoom microscope (Leica MZ6, Leica Microsystems GmbH) using a scalpel 28 d after inoculation. Genomic DNA of plant material was isolated wth the DNeasy Plant Mini Kit (Qiagen). Genomic DNA (1.1 µg) was sonicated to approximately 600 bp fragments using a Bioruptor (Diagenode), heated for 10 min at 99 °C and immediately cooled on ice for 10 min. 100 ng DNA were removed and used as input samples for array hybridization. Immunoprecipitation was performed by incubating 1 µg of sonicated DNA with 10 µg of 5-mC monoclonal antibody (Diagenode) in 600 µl IP-Buffer (10 mM Na-Phosphate Buffer, 0.14 M NaCl, 0.05 % Triton X-100, pH 7.0) at 4°C for 12 h. After addition of 100 µl of Dynabeads Protein G (Life Technologies) and incubation at 4°C for 3 h, Dynabeads were washed twice with 600 µl IP-Buffer for 10 min. DNA elution was performed by vortexing the dynabeads three times in 200 µl TE buffer with increasing SDS-concentrations (0.1%, 0.5%, 1.5%). DNA was purified by phenol-chloroform extraction and ethanol precipitation. Input DNA and mCIP eluate were processed with the Whole Genome Amplification kit (Sigma) until a DNA yield of >7 µg was reached.
|
| Label |
biotin
|
| Label protocol |
Samples (approximately 7 µg of amplified DNA each) were fragmented and terminally labeled with biotin using the GeneChip Mapping 10K Xba kit (Affymetrix).
|
| |
|
| Channel 2 |
| Source name |
Input DNA from A. thaliana crown galls
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: crown galls from Agrobacterium tumefaciens strain C58noc inoculated inflorescence stalks
antibody: none (input)
|
| Growth protocol |
Arabidopsis plants were cultivated in growth chambers at 22°C during the light and 16°C during the dark period in 12 h intervals. Arabidopsis plants were cultivated in growth chambers at 22°C during the light and 16°C during the dark period in 12 h intervals.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tissue was separated from the host inflorescence stalk under a stereozoom microscope (Leica MZ6, Leica Microsystems GmbH) using a scalpel 28 d after inoculation. Genomic DNA of plant material was isolated wth the DNeasy Plant Mini Kit (Qiagen). Genomic DNA (1.1 µg) was sonicated to approximately 600 bp fragments using a Bioruptor (Diagenode), heated for 10 min at 99 °C and immediately cooled on ice for 10 min. 100 ng DNA were removed and used as input samples for array hybridization. Immunoprecipitation was performed by incubating 1 µg of sonicated DNA with 10 µg of 5-mC monoclonal antibody (Diagenode) in 600 µl IP-Buffer (10 mM Na-Phosphate Buffer, 0.14 M NaCl, 0.05 % Triton X-100, pH 7.0) at 4°C for 12 h. After addition of 100 µl of Dynabeads Protein G (Life Technologies) and incubation at 4°C for 3 h, Dynabeads were washed twice with 600 µl IP-Buffer for 10 min. DNA elution was performed by vortexing the dynabeads three times in 200 µl TE buffer with increasing SDS-concentrations (0.1%, 0.5%, 1.5%). DNA was purified by phenol-chloroform extraction and ethanol precipitation. Input DNA and mCIP eluate were processed with the Whole Genome Amplification kit (Sigma) until a DNA yield of >7 µg was reached.
|
| Label |
biotin
|
| Label protocol |
Samples (approximately 7 µg of amplified DNA each) were fragmented and terminally labeled with biotin using the GeneChip Mapping 10K Xba kit (Affymetrix).
|
| |
|
| |
| Hybridization protocol |
Approximately 7 µg of amplified and terminally labeled DNA were hybridized to tiling arrays using the GeneChip HWS kit (Affymetrix). Array hybridization was carried out at 45°C at 60 rpm for 16 h in an Affymetrix hybridization oven. Prior to scanning, arrays were washed in the Fluidics Station (Affymetrix) using script FS450_0001.
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000.
|
| Description |
A. thaliana tumor mCIP enrichment, biological replicate 3
|
| Data processing |
Data from mCIP and input pairs were loess normalized; log2(mCIP/input) signal log ratios (SLR) were reported in bedGraph files. Coordinates of genomic probes were mapped to the A. thaliana TAIR9 sequence assembly. Analyses were performed in R (http://www.r-project.org) with the Bioconductor package Starr (http://www.bioconductor.org).
Additional results files: K2_K3_K4_smoothed.bedGraph; T2_T3_T4_smoothed.bedGraph; unchanged_regions.bed; hypomethylated_regions.bed; hypermethylated_regions.bed
Results file descriptions: SLRs were quantiles normalized and subjected to a 500 bp sliding window median smoothing across biological replicates of the two experimental groups. The smoothed SLRs were reported in files K2_K3_K4_smoothed.bedGraph and T2_T3_T4_smoothed.bedGraph. The CMARRT algorithm was employed to define mCIP-enriched genomic regions in smoothed SLRs of the experimental groups. File unchanged_regions.bed reports enriched regions present in both groups, hypomethylated_regions.bed reports regions enriched only in tumor-free stalk tissue and hypermethylated_regions.bed reports regions enriched only in crown galls; these regions are discussed in the paper. Analyses were performed in R (http://www.r-project.org) with the Bioconductor package Starr (http://www.bioconductor.org).
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| |
|
| Submission date |
May 01, 2012 |
| Last update date |
Feb 19, 2013 |
| Contact name |
Claus Juergen Scholz |
| Organization name |
University Hospital Würzburg
|
| Department |
Core Unit SysMed
|
| Street address |
Josef-Schneider-Str. 2
|
| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10977 |
| Series (1) |
| GSE37680 |
Genome-wide analysis of the cytosine methylation profile in Arabidopsis thaliana crown galls |
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