|
| Status |
Public on Jan 07, 2016 |
| Title |
Cold5 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
cold r5 input
|
| Organism |
Zea mays |
| Characteristics |
cultivar: B73
replication: 5
tissue: flag leaf tissue (mature)
|
| Growth protocol |
seeds grown for 14 days under standard conditions. Seedlings transferred to cold room (4C) for four hours and returned. Stress treatment was performed every other day for four treatments. Plants were subsequently moved to a greenhouse to grow to maturity under standard conditions.
|
| 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 |
cold r5 IP
|
| Organism |
Zea mays |
| Characteristics |
cultivar: B73
replication: 5
tissue: flag leaf tissue (mature)
|
| Growth protocol |
seeds grown for 14 days under standard conditions. Seedlings transferred to cold room (4C) for four hours and returned. Stress treatment was performed every other day for four treatments. Plants were subsequently moved to a greenhouse to grow to maturity under standard conditions.
|
| 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 User Guide protocol, which specifies parameters for the MS2000 Scanner (Nimblegen) used to collect data.
|
| Data processing |
Images were aligned and quantified using NimbleScan software (Roche NimbleGen) which produced .pair reports of raw data.
Raw data (pair) files were exported from NimbleScan into the Bioconductor statistical environment in R (Gentleman et al., 2004). For all samples methylated DNA immunoprecipitation (MeDIP) enrichments were estimated or each probe in a linear model accounting for array, dye, and sample effects using the limma package (Smyth, 2004). Statistical contrasts were then fit between IP samples and genomic DNA control samples (input) for each sample. Moderated t-statistics and the log-odds score for differential MeDIP enrichment was computed by empirical Bayes shrinkage of the standard errors with the false discovery rate controlled to 0.05.
|
| |
|
| Submission date |
Jan 24, 2015 |
| Last update date |
Jan 07, 2016 |
| 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) |
| GSE65266 |
Limited evidence for consistent changes in maize DNA methylation patterns following environmental stress. |
|
