tissue: whole rosettes genotype: Van0 time: end of the day (ED; ZT8)
Treatment protocol
For each accession, 5 rosettes from 5 week old plants were harvested and pooled in the last hour of the day (ED; ZT8), or the last hour of the night (EN; ZT24), or 6h into an extended night (XN; ZT30).
Growth protocol
20 A.th. accessions were grown on soil in a 8h light / 16h dark cycle, at a light intensity of 160 µmol m-2 s-1 and a constant temperature of 20°C for 5 weeks.
Extracted molecule
total RNA
Extraction protocol
Material from a pool of 5 rosettes was snap frozen in liquid nitrogen and stored until further use at -80C or was processed directly as follows. Frozen plant material was homogenized and total RNA was extracted from 50 mg of homogenized plant material using Qiagen RNeasy Plant mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Before and after DNaseI digestion (Turbo DNA-free DNase I; Applied Biosystems/Ambion, Darmstadt, Germany), RNA concentration and integrity were measured using a NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, USA) and an Agilent-2100 Bioanalyzer using RNA 6000 NanoChips (Agilent Technologies, Böblingen, Germany).
Label
biotin
Label protocol
Approximately 10 µg of total RNA was processed to produce biotinylated cRNA targets.
Hybridization protocol
standard Affymetrix procedures
Scan protocol
standard Affymetrix procedures
Data processing
Background correction and quantile normalization were performed using the ‘preprocessCore’ and ‘affyPLM’ packages (Bioconductor; Gentleman et al. in Genome Biology 2004, 5(10): R80) to calculate RMA signal intensities. To identify probes potentially affected by SFPs in the raw data, a combination of a median polish method, adapted from (Xie et al. in Theoretical and Applied Genetics 2009, 119(1): 151-164), and a one-way ANOVA were used to select individual probes that were significantly altered between accessions (p < 0.01). To identify probes, which showed lower probe signal intensity in an accession compared to Col-0, significant contrasts were subsequently identified in a post-hoc Tukey HSD test (p < 0.01). The list of 21,297 probes with significant signal alterations in any of the accessions compared to Col-0, was combined with 22,297 Cvi sequence SNPs available from the 1,001 genome project. Additional SNPs were added by taking available data for 19 of the 20 accessions (except for Bu-2, Bu-0 used instead) derived from genotyping accessions using a custom Affymetrix SNP chip containing 250K SNPs (Atwell et al. in Nature 2010, 465(7298): 627-631). The final list of potentially SNP containing probes comprised 44,863 SNPs. Joint masking of all these probes led to a removal of 1,481 probe sets that had <6 probes remaining, reducing the number of probe sets to a final number of 21,329.
Submission date
Jul 21, 2015
Last update date
Dec 31, 2023
Contact name
Eva-Theresa Pyl
Organization name
Max Planck Institute of Molecular Plant Physiology
