|
| Status |
Public on Aug 12, 2013 |
| Title |
TIL09 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
til09 input
|
| Organism |
Zea mays |
| Characteristics |
genotype: til09
tissue: seedling leaf (L3)
dna: input control
|
| Treatment protocol |
genomic DNAs were sonicated, amplified, and fluorophore-labeled
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNAs were extracted using CTAB and purified using phenol:chloroform/Na-Acetate purification and EtOH precipitation. 15-30ug of genomic DNA were sonicated (5 reps x 10 seconds with >1 minute on ice between reps). Methylated DNAs were isolated from 400ng of sonicated DNA using the Zymo Research Methylated DNA IP kit (Cat # D5101) which contains the anti-5-methylcytosine monoclonal antibody. 50-100ng of methylated DNA and 50-100ng of sonicated (input control) DNAs were amplified using the Sigma Whole Genome Amplification kit (Cat # WGA2-10RXN).
|
| Label |
Cy3
|
| Label protocol |
Amplified input and methylated DNA samples were labeled (3 x 1ug per reaction), hybridized to the array and washed according to the array manufacturers protocol. Input DNAs were labeled using Cy3 and the immunoprecipitated methylated DNAs (IP) were labeled with Cy5.
|
| |
|
| Channel 2 |
| Source name |
til09 IP
|
| Organism |
Zea mays |
| Characteristics |
genotype: til09
tissue: seedling leaf (L3)
antibody: anti-5-methylcytosine monoclonal antibody (Zymo, Cat # D5101)
dna: methylated DNA
|
| Treatment protocol |
genomic DNAs were sonicated, methylated DNAs were isolated, amplified, and fluorophore-labeled
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNAs were extracted using CTAB and purified using phenol:chloroform/Na-Acetate purification and EtOH precipitation. 15-30ug of genomic DNA were sonicated (5 reps x 10 seconds with >1 minute on ice between reps). Methylated DNAs were isolated from 400ng of sonicated DNA using the Zymo Research Methylated DNA IP kit (Cat # D5101) which contains the anti-5-methylcytosine monoclonal antibody. 50-100ng of methylated DNA and 50-100ng of sonicated (input control) DNAs were amplified using the Sigma Whole Genome Amplification kit (Cat # WGA2-10RXN).
|
| Label |
Cy5
|
| Label protocol |
Amplified input and methylated DNA samples were labeled (3 x 1ug per reaction), hybridized to the array and washed according to the array manufacturers protocol. Input DNAs were labeled using Cy3 and the immunoprecipitated methylated DNAs (IP) were labeled with Cy5.
|
| |
|
| |
| Hybridization protocol |
24-34ug of labeled DNAs (input DNA, IP DNA) were hybridized to the arrays according to the array manufacturer protocol (42°C, 16-20hrs).
|
| Scan protocol |
Arrays were scanned according to the NimbleScan CGH User Guide protocol, which specifies parameters for the GenePix4000B Scanner (Axon/Molecular Devices) or MS2000 Scanner (Nimblegen) used to collect data.
|
| Description |
This Sample represents up to 3 replicates.
|
| Data processing |
Images were aligned and quantified using NimbleScan software (Roche NimbleGen) which produced .pair reports of raw data.
Pair files for 2.1M and 1.4M platforms that were exported from NimbleScan were imported into the Bioconductor statistical environment (Gentleman et al., 2004). 2.1M platform pair files were subset to the 1.4M platform probes to facilitate combined analysis. For each genotype, designated as HapMap2 ‘HM’ lines (excluding B73), microarray data channels were assigned the following factors: B73, HM_line, B73 input, or HM_line input depending on sample derivation. For samples in which three replicates were not available, individual replicates were duplicated to maintain normalization methods across all samples. Non-maize probes and vendor-supplied process control probes were configured to have analytical weights of zero. Variance-stabilizing normalization was used to account for array-specific effects. Factor-specific hybridization coefficients were estimated by fitting fixed linear model accounting for dye and sample effects to the data using the limma package (Smyth 2004). To compute biologically relevant information about each HM line’s DNA methylation, the following contrasts were then computed: B73 IP vs B73 input (B73 methylation); HM_line input vs B73 input (CGH and differential hybridization efficiency); HM_line IP vs [HM_line input vs B73 input] (HM_line methylation corrected for differential hybridization efficiency); B73 IP vs HM_line IP vs [HM_line input vs B73 input]] (differential DNA methylation corrected for differential hybridization efficiency). Moderated t-statistics and the log-odds score for differential MeDIP enrichment were computed by empirical Bayes shrinkage of the standard errors with the False Discovery Rate controlled to 0.05 (Smyth 2004).
|
| |
|
| Submission date |
May 15, 2013 |
| Last update date |
Aug 13, 2013 |
| Contact name |
Steve R Eichten |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Minnesota
|
| Department |
Plant Biology
|
| Lab |
Springer Lab
|
| Street address |
1445 Gortner Ave
|
| City |
St. Paul |
| State/province |
MN |
| ZIP/Postal code |
55108 |
| Country |
USA |
| |
|
| Platform ID |
GPL15621 |
| Series (1) |
| GSE46949 |
Epigenetic and genetic influences on DNA methylation variation in maize populations |
|
