|
| Status |
Public on Feb 06, 2011 |
| Title |
CDC6 samples vs INPUT (genomic DNA) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ChIP: genomic DNA enriched in CDC6 binding fragments
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
sample type: ChIP fraction
antibody: anti-HA (A2095; SIGMA)
|
| Treatment protocol |
ChIP assays were carried out using 10-day-old plants except that they were preincubated with 50µM MG132 prior to the fixation step. For immunoprecipitation, 10µl of anti-Myc (SC-40ac; Santa Cruz Biotechnology) or anti-HA (A2095; SIGMA), were incubated with 1mg of chromatin extract, previously sonicated, to obtain DNA fragments of ~500-1000bp.
|
| Growth protocol |
Arabidopsis seedlings (Col-0 ecotype) were grown in MS salts medium supplemented with 1% sucrose and 1% agar in a 16h/8h light/dark regime at 22°C. Plants constitutively expressing the Myc-ORC1a-tagged and the hemagglutinin (HA)-tagged Arabidopsis CDC6a protein have been described elsewhere (Castellano et al., 2001; Sanchez and Gutierrez, 2009).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After washing, the immunocomplexes were eluted and incubated at 65ºC for 6h to reverse crosslinking. Residual protein was eliminated with 20µg of proteinase K and phenol-chloroform extractions. The DNA obtained (ChIP and input) was cleaned up using Affymetrix cDNA cleanup columns.
|
| Label |
biotin
|
| Label protocol |
DNA was amplified using Affymetrix Chromatin Immunoprecipitation Assay protocol. 7.5 μg of amplification product were fragmented and labeled using GeneChip® WT Double-stranded DNA terminal labeling kit (Affymetrix).
|
| |
|
| Channel 2 |
| Source name |
INPUT: genomic DNAs
|
| Organism |
Arabidopsis thaliana |
| Characteristics |
sample type: input
antibody: none
|
| Treatment protocol |
ChIP assays were carried out using 10-day-old plants except that they were preincubated with 50µM MG132 prior to the fixation step. For immunoprecipitation, 10µl of anti-Myc (SC-40ac; Santa Cruz Biotechnology) or anti-HA (A2095; SIGMA), were incubated with 1mg of chromatin extract, previously sonicated, to obtain DNA fragments of ~500-1000bp.
|
| Growth protocol |
Arabidopsis seedlings (Col-0 ecotype) were grown in MS salts medium supplemented with 1% sucrose and 1% agar in a 16h/8h light/dark regime at 22°C. Plants constitutively expressing the Myc-ORC1a-tagged and the hemagglutinin (HA)-tagged Arabidopsis CDC6a protein have been described elsewhere (Castellano et al., 2001; Sanchez and Gutierrez, 2009).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After washing, the immunocomplexes were eluted and incubated at 65ºC for 6h to reverse crosslinking. Residual protein was eliminated with 20µg of proteinase K and phenol-chloroform extractions. The DNA obtained (ChIP and input) was cleaned up using Affymetrix cDNA cleanup columns.
|
| Label |
biotin
|
| Label protocol |
DNA was amplified using Affymetrix Chromatin Immunoprecipitation Assay protocol. 7.5 μg of amplification product were fragmented and labeled using GeneChip® WT Double-stranded DNA terminal labeling kit (Affymetrix).
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed by the standard Affymetrix protocol.
|
| Scan protocol |
Scanning was performed at 0.7μm resolution using a GeneChip Scanner 3000 7G (Affymetrix Inc, Santa Clara, CA).
|
| Description |
CDC6 x2 replicates vs INPUT x 3 replicates.
CDC6 ChIP replicates:
CDC6_3.CEL
CDC6_4.CEL
INPUT replicates:
Wt_1_At35b_MR_v04.CEL
Wt_2_At35b_MR_v04.CEL
Wt_3_At35b_MR_v04.CEL
Arabidopsis thaliana genome tiling array used in this study has 2.8 million probes, spaced 35 bases apart, with one 25 base probe within each window. Start of Probe position on chromosome based on TAIR7 genome build (probe length is 25 nts and positions are 0-based not 1-based). Tiles most of the Arabidopsis genome.
|
| Data processing |
ChIP data were quantile normalized to genomic DNA (INPUT) using Affymetrix Tiling Analysis Software. Each data set was then normalized so that the mean signal across all probes in the genome was zero.
Results file (found on Series record): Enrichment ratio calculated from normalized intensities and averaged over all replicates. Positive values indicate enrichment of the ChIP fraction versus the INPUT sample.
|
| |
|
| Submission date |
May 20, 2010 |
| Last update date |
Feb 06, 2011 |
| Contact name |
Juan Carlos Oliveros |
| Organization name |
CNB, CSIC
|
| Street address |
Darwin 3
|
| City |
Cantoblanco |
| State/province |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
| |
|
| Platform ID |
GPL10977 |
| Series (1) |
| GSE21928 |
Mapping replication-initiation proteins ORC1 and CDC6 in Arabidopsis thaliana |
|
