|
| Status |
Public on Mar 24, 2015 |
| Title |
R1_3.7.3_3rdleaf |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
R1.3.7.3_input, R1
|
| Organism |
Zea mays |
| Characteristics |
line: A188
tissue: third seedling leaf
replication: 1
sample type: input
|
| Treatment protocol |
Genomic DNAs were sonicated, amplified, and fluorophore-labeled.
|
| Growth protocol |
Maize plants (A188 genotype) were grown under controlled conditions in a greenhouse at the University of Wisconsin-Madison (Madison, WI) with a light cycle of 15 hours lights on and 9 hours lights off each day. Maize plants were watered daily as needed.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Callus used for DNA extraction was collected from each cell line prior to plating onto R1 media after six months of culturing. The uppermost flag leaf of R0 plants and the 3rd leaf of R1 plants were harvested for DNA extraction to conduct meDIP-Chip profiling (Zymo Research DNA methylation IP kit D5101). All tissues were immediately flash-frozen in liquid N2. DNA was isolated using a modified CTAB method.
|
| 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 manufacturer's protocol. Input DNAs were labeled using Cy3 and the immunoprecipitated methylated DNAs (IP) were labeled with Cy5.
|
| |
|
| Channel 2 |
| Source name |
R1.3.7.3_IP, R1
|
| Organism |
Zea mays |
| Characteristics |
line: A188
tissue: third seedling leaf
replication: 1
sample type: methylated DNA (IP pulldown)
|
| Treatment protocol |
Genomic DNAs were sonicated, methylated DNAs were isolated, amplified, and fluorophore-labeled.
|
| Growth protocol |
Maize plants (A188 genotype) were grown under controlled conditions in a greenhouse at the University of Wisconsin-Madison (Madison, WI) with a light cycle of 15 hours lights on and 9 hours lights off each day. Maize plants were watered daily as needed.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Callus used for DNA extraction was collected from each cell line prior to plating onto R1 media after six months of culturing. The uppermost flag leaf of R0 plants and the 3rd leaf of R1 plants were harvested for DNA extraction to conduct meDIP-Chip profiling (Zymo Research DNA methylation IP kit D5101). All tissues were immediately flash-frozen in liquid N2. DNA was isolated using a modified CTAB method.
|
| 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 manufacturer's 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's protocol (42°C, 16-20hrs).
|
| Scan protocol |
Arrays were scanned according to the NimbleScan CGH User Guide protocol, which specifies parameters for the MS2000 Scanner (NimbleGen) used to collect data.
|
| Description |
R1.3.7.3
|
| Data processing |
Images were aligned and quantified using NimbleScan software (Roche NimbleGen) which produced .pair reports of raw data.
Pair files that were exported from NimbleScan were imported into the Bioconductor statistical environment (Gentleman et al., 2004). Signal was first loess normalized within each array to minimize space effect and then quantile normalized across arrays to minimize batch effect. This was done through the Ringo Bioconductor package in R for the management and normalization of NimbleGen microarray data.
|
| |
|
| Submission date |
Apr 02, 2014 |
| Last update date |
Mar 24, 2015 |
| 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) |
| GSE56479 |
Consistent and Heritable Alterations of DNA Methylation are Induced by Tissue Culture in Maize |
|
