genotype: abi2-1, erecta homozygous, hlq/+ heterozygous and +/+ wild type
Treatment protocol
samples were not treated.
Growth protocol
~1,000 Arabidopsis thaliana seeds from a self-fertilization of heterozygote for the mutant hlq gene in the genetic background abi2-1/abi2-1 homozygous (Landsberg erecta ecotype) were soaked in water and kept in the dark at 4°C for 3 days before being placed in rows and germinated on Petri plates containing 0.5x Murashige & Skoog salts (Research Products International, Mt. Prospect, IL), 1% sucrose, 0.5% phytagel (SIGMA-ALDRICH, St. Louis, MO). Plates were kept vertically under continuous light (~ 100 µE/m-2s-1) at 21°C for two weeks. Then seedlings were collected in microfuge tubes, frozen in liquid nitrogen and stored at -80°C before RNA extraction. The accessions used in this study are listed as follows: Col-0 (stock # CS60000), abi2-1 (CS23), Landsberg erecta (Ler) (CS20), available from the Arabidopsis Biological Resource Center (Ohio State University; http://abrc.osu.edu/).
Extracted molecule
total RNA
Extraction protocol
For each individual pool, 100mg sample (~40 seedlings) was ground in a mortar with liquid nitrogen. Total RNA was extracted by using Trizol (Invitrogen, Carlsbad, CA) according to manufacturer's guidelines. Nucleotide pellet was washed with 70% EtOH twice and dried in air. After dissolving pellet in DEPC-treated water, the RNA concentration was measured by Nano Drop and the final concentration was adjusted to 1 µg/µL with DEPC water.
Label
Cy3
Label protocol
~2.5 µg of total RNA was used per sample per labeling reaction for reverse transcription into cDNA. Strand-specific cRNA was labeled with Amino Allyl MessageAmp aRNA Amplification Kit (Ambion; cat#: AM1753) according to the manufacturer's protocol (Grand Island, NY, USA). Each sample was run on a Bioanalyzer (Agilent Technologies, Santa Clara, CA) before and after labeling.
~1,000 Arabidopsis thaliana seeds from a self-fertilization of heterozygote for the mutant hlq gene in the genetic background abi2-1/abi2-1 homozygous (Landsberg erecta ecotype) were soaked in water and kept in the dark at 4°C for 3 days before being placed in rows and germinated on Petri plates containing 0.5x Murashige & Skoog salts (Research Products International, Mt. Prospect, IL), 1% sucrose, 0.5% phytagel (SIGMA-ALDRICH, St. Louis, MO). Plates were kept vertically under continuous light (~ 100 µE/m-2s-1) at 21°C for two weeks. Then seedlings were collected in microfuge tubes, frozen in liquid nitrogen and stored at -80°C before RNA extraction. The accessions used in this study are listed as follows: Col-0 (stock # CS60000), abi2-1 (CS23), Landsberg erecta (Ler) (CS20), available from the Arabidopsis Biological Resource Center (Ohio State University; http://abrc.osu.edu/).
Extracted molecule
total RNA
Extraction protocol
For each individual pool, 100mg sample (~40 seedlings) was ground in a mortar with liquid nitrogen. Total RNA was extracted by using Trizol (Invitrogen, Carlsbad, CA) according to manufacturer's guidelines. Nucleotide pellet was washed with 70% EtOH twice and dried in air. After dissolving pellet in DEPC-treated water, the RNA concentration was measured by Nano Drop and the final concentration was adjusted to 1 µg/µL with DEPC water.
Label
Cy5
Label protocol
~2.5 µg of total RNA was used per sample per labeling reaction for reverse transcription into cDNA. Strand-specific cRNA was labeled with Amino Allyl MessageAmp aRNA Amplification Kit (Ambion; cat#: AM1753) according to the manufacturer's protocol (Grand Island, NY, USA). Each sample was run on a Bioanalyzer (Agilent Technologies, Santa Clara, CA) before and after labeling.
Hybridization protocol
A pair of Cy5- and Cy3-labeled samples were mixed in equimolar proportions (~1000 ng per sample) and hybridized. Conditions of hybridization and washing were according to the protocol published by Galbraith DW, Janda J, Lambert GM: Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type. In: Methods in Molecular Biology: Flow Cytometry Protocols. vol. 699; 2011: 407-429.
Scan protocol
GenePix Autoloader 4200AL Scanner (Molecular Devices, Sunnyvale, CA) with laser excitation at 532 and 635 nm at the resolution of ten pixels per micron, and saved as 16-bit grayscale multi-image TIFF files.
Intensity values were extracted using GenePix Pro 6.0 (Molecular Devices, Sunnyvale, CA) and saved as gpr and txt files for each block individually. The data for each array were lowess normalized followed by ANOVA analysis of differential gene expression using empirical Bayes methods to moderate the standard deviations between genes. This was done by the software package Linear Models for Microarray Data (limma) (Smyth, G.K., Michaud, J., and Scott, H.S. (2005). Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 21, 2067-2075) written in R language under the BioConductor platform (Gentleman, R., Carey, V., Bates, D., Bolstad, B., Dettling, M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., Hornik, K., Hothorn, T., Huber, W., Iacus, S., Irizarry, R., Leisch, F., Li, C., Maechler, M., Rossini, A., Sawitzki, G., Smith, C., Smyth, G., Tierney, L., Yang, J., and Zhang, J. (2004). Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 5, R80) version R2.5.1 (http://www.r-project.org/). Normalization “within” and “between arrays” scaled the log2-ratios of each two color experiment to have the same median-absolute-deviation across arrays. We calculated a dye effect coefficient in addition to the genotype coefficient and signal amplitude across all probes and samples.
