|
| Status |
Public on Nov 26, 2012 |
| Title |
mop1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
mop1 input
|
| Organism |
Zea mays |
| Characteristics |
genotype: B73
replication: 1
tissue: 14 day whole seedling
dna: input
|
| Treatment protocol |
sonicated, amplified gDNA control
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using the CTAB method (Doyle 1987). 5-10ug of gDNA in 650-700uL nuclease-free water was sonicated for five, ten- second pulses as per the methods of Haun et al., 2008. Samples were quantified and run on 1.5% agarose gels to verify that DNAs were fragmented to 100-500bp. Methylated DNA was immunoprecipitated with an anti-5-methylcytosine monoclonal antibody from 400ng sonicated DNA using the Methylated DNA IP Kit (Zymo Research, Orange, CA; Cat # D5101).
|
| Label |
Cy3
|
| Label protocol |
For each amplified IP and input sample, 3 ug of amplified DNA were labeled using the Dual-Color Labeling Kit (Roche NimbleGen, Cat # 05223547001) according to the array manufacturer’s protocol (Roche NimbleGen Methylation User- Guide v7.0). Each IP sample was labeled with Cy5 and each input/control sonicated DNA was labeled with Cy3.
|
| |
|
| Channel 2 |
| Source name |
mop1 IP
|
| Organism |
Zea mays |
| Characteristics |
genotype: B73
replication: 1
tissue: 14 day whole seedling
dna: 5mc enriched DNA
|
| Treatment protocol |
sonicated, amplified, meDIP DNA
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated using the CTAB method (Doyle 1987). 5-10ug of gDNA in 650-700uL nuclease-free water was sonicated for five, ten- second pulses as per the methods of Haun et al., 2008. Samples were quantified and run on 1.5% agarose gels to verify that DNAs were fragmented to 100-500bp. Methylated DNA was immunoprecipitated with an anti-5-methylcytosine monoclonal antibody from 400ng sonicated DNA using the Methylated DNA IP Kit (Zymo Research, Orange, CA; Cat # D5101).
|
| Label |
Cy5
|
| Label protocol |
For each amplified IP and input sample, 3 ug of amplified DNA were labeled using the Dual-Color Labeling Kit (Roche NimbleGen, Cat # 05223547001) according to the array manufacturer’s protocol (Roche NimbleGen Methylation User- Guide v7.0). Each IP sample was labeled with Cy5 and each input/control sonicated DNA was labeled with Cy3.
|
| |
|
| |
| Hybridization protocol |
Samples were hybridized to the custom 3x1.4M probe array for 16–20 hrs at 42C.
|
| Scan protocol |
Slides were washed and scanned according to NimbleGen’s protocol using the MS200 microarray scanner. Images were aligned and quantified using NimbleScan software (Roche NimbleGen) producing raw data reports for each probe on the array.
|
| Data processing |
The resulting microarray data were imported into the Bioconductor statistical environment (http://bioconductor.org/). 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 [55].
|
| |
|
| Submission date |
Jul 18, 2012 |
| Last update date |
Nov 27, 2012 |
| 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) |
| GSE39460 |
Spreading of heterochromatin is limited to specific families of maize retrotransposons |
|
