Material was derived from inflorescences of synchronized 35S:AP1-GR ap1 cal plants.
Extracted molecule
other
Extraction protocol
Decapped mRNA was isolated from poly(A)+ mRNA using a modified RLM-RACE protocol.
Label
Cy5
Label protocol
In brief, first and second strand cDNA was synthesized using a poly(A)-primer with a T7 promoter sequence. Then, in vitro transcription was performed using the Megascript T7 kit (Ambion, Austin, TX). For the oligonucleotide array, dye molecules were coupled to the amplified RNA, and the dye-labeled RNA was fragmented before hybridization.
Material was derived from inflorescences of synchronized 35S:AP1-GR ap1 cal plants.
Extracted molecule
polyA RNA
Extraction protocol
PolyA RNA was isolated using the Micro-FastTrack 2.0 mRNA isolation kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Label
Cy3
Label protocol
protocol Dye-labeled antisense RNA was previously described (Wellmer et al. (2006) PLoS Genetics 2, e117). In brief, first and second strand cDNA was synthesized using a poly(A)-primer with a T7 promoter sequence. Then, in vitro transcription was performed using the Megascript T7 kit (Ambion, Austin, TX). For the oligonucleotide array, dye molecules were coupled to the amplified RNA, and the dye-labeled RNA was fragmented before hybridization.
Hybridization protocol
Dye-labeled antisense RNA was hybridized to microarrays using a MAUI hybridization system (BioMicro Systems, Salt Lake City, UT, USA) as previously described (Wellmer et al. (2006) PLoS Genetics 2, e117). Specifically, hybridizations were done as follows: dye-labeled antisense RNA preparations were dried down and the resulting pellets were re-suspended in 5 ul 10 mM EDTA and 45 ul SlideHyb Buffer #1 (Ambion, Austin, Texas, United States) and hybridized for 14 h to microarrays at 48C using a MAUI hybridization system (BioMicro Systems, Salt Lake City, Utah, United States) according to the manufacturer's instructions.
Scan protocol
Microarrays were scanned and the data acquired with an Axon GenePix 4200A scanner using GenePix v5.0 analysis software.
Description
Plants were grown on a soil:vermiculite:perlite mixture under constant illumination at 20°C. Immediately after the onset of bolting, the inflorescences of 35S:AP1-GR ap1 cal plants were treated with a solution containing 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 0.01% (v/v) ethanol, and 0.015% (v/v) Silwet L-77. For each time point, tissue from ~25 plants was collected using jewelers forceps. Tissue was removed as close to the surface of the inflorescence as possible to ensure an enrichment of meristematic cells.
Data processing
Data were processed in eCADS (Dabney and Storey (2007) Genome Biol. 8, R44).
