ecotype: Col-0 tissue: seed developmental stage according to boyes et al. (2004): 11-13 DAP chip antibody: anti-ABI3 Antibody
Growth protocol
Arabidopsis plants were grown on soil, with a 16 h light/ 8 h dark cycle
Extracted molecule
genomic DNA
Extraction protocol
Chromatin was isolated from unripe seeds of Arabidopsis thaliana Col-0, harvested 11-13 days after pollination. Cross-linking was done with 1% formaldehyde for 20 min under vacuum, terminated by addition of glycine with a final concentration of 125 mM. Seeds were Potter-homogenized, filtered and the nuclei collected by centrifugation. Nuclear membranes were dissolved with detergents and the chromatin was collected by high speed centrifugation. Chromatin was fragmented to an average size of 500 bp by ultra sonic treatment. 10 µg chromatin were combined with 2 µg anti-ABI3 antibody. The antibody-chromatin complex was captured by protein A coated magnetic beads (Invitrogen). After different washings the TF-DNA complex was eluted from the beads at 65°C for 15 min using 1% SDS. Eluates were incubated overnight at 65°C to reverse the cross-links. DNA fragments were purified using a QIAquick PCR Purification Kit (Qiagen) and their concentration was determined with a NanoDrop 3300 (Thermo Fisher Scientific) fluorescence spectrophotometer. An aliquot (0.5 µg) of non-immunoprecipitated chromatin served as input control. DNA fragments were blunted and ligated with the adapters oJW102 and oJW103 (Young lab:jura.wi.mit.edu/young_public/hESregulation/ChIP.html). Adapter-ligated fragments were amplified using oJW102 as sole primer.
Label
[α-33P]dCTP
Label protocol
Labelled probes were obtained by combining three parallel random priming reactions using the Megaprime Kit (GE Healthcare, Uppsala, Sweden), 1ng amplified chromatin, and 50µCi [α-33P]dCTP per reaction.
Hybridization protocol
Arrays were prehybridized for 1h at 65°C in Church buffer (0,5M sodium phosphate, pH7,2, 7% SDS, 1% BSA, 1mM EDTA, 10µg/ml sheared salmon sperm DNA) per cm2 of membrane in a rotating thermal oven. Labelled probes were added and hybridization continued for 4 days. Membranes were washed twice in 0,2x SSC, 0,1% SDS for 20 min at 65°C and then placed on a moist Whatman paper, wrapped in Saran wrap and exposed to a Fuji image plate for 10 days in a lead chamber.
Scan protocol
Signals were detected using a FLA 3000 phosphoimager at 50µm resolution and 16 bit grey scale (Fuji). Spot intensities were obtained by aligning the pre-defined spotting pattern using ArrayVision software (GE Healthcare).
Description
070420ABI3 Raw data file: 070420_B_ABI3.txt ChIP technical replicate of biol. replicate 3 Hybridization on membrane B
Data processing
Five ABI3 ChIP-chip experiments (three biological and two technical replicates) were performed, each consisting of a pair of hybridisations with amplified input chromatin and immuno-precipitated chromatin. For normalization, the median of log2 transformed signal intensities of each hybridisation was centred at zero. Quantile normalization (Bolstad et al. 2003) was applied separately to datasets of input and immuno-precipitated chromatin. Duplicated spotting of each amplicon on the arrays provided an internal quality control, which was used to exclude all signals with a difference of normalized log2 intensities larger than one between the two spots (1.56% of all signals). Missing values were again substituted by the mean of normalized log2 intensities from other hybridisations of the same kind, chromatin or immuno-precipitated chromatin, if the signal was excluded in no more than two hybridisations. Finally, the mean of normalized log2 intensities from both spots of each amplicon was calculated and the mean values of hybridisations with input chromatin were subtracted from the mean values of hybridizations with immuno-precipitated chromatin, resulting in log2 enrichment factors for most (> 99.85%) of the spotted amplicons in all five experiments.
