Affymetrix GeneChip Arabidopsis Genome [ATH1-121501]

CONSTITUTIVELY PHOTOMORPHOGENIC1 Is Required for the UV-B Response in Arabidopsis.

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Seven-day-old white-light-grown wild-type, cop1-4 or hy5-1 mutant Arabidopsis seedlings were exposed for fifteen minutes to polychromatic radiation with decreasing short-wave cut-off in the UV range (WG305 = +UV-B, WG327 = -UV-B) and samples were taken 1 h after the onset of irradiation.

genotype

strain

UV-B wavelength

Transcription profiling of Arabidopsis wild type, cop1-4, hy5-1 mutant seedlings exposed to polychromatic radiation

Seven-day-old white-light-grown wild-type, cop1-4 or hy5-1 mutant Arabidopsis seedlings were exposed for fifteen minutes to polychromatic radiation with decreasing short-wave cut-off in the UV range (WG305 = +UV-B, WG327 = -UV-B) and samples were taken 1 h after the onset of irradiation.


Affymetrix:Protocol:ExpressionStat

Title: Affymetrix CHP Analysis (ExpressionStat). Description:

Affymetrix:Protocol:Hybridization-EukGE-WS2v4

Title: Fluidics Station Protocol. Description:

P-AFFY-6

Title: Affymetrix CEL analysis. Description:

P-MEXP-9939

Arabidopsis seeds were surface-sterilized with sodium-hypochlorite, plated on Murashige and Skoog medium (MS, Duchefa) containing 1% sucrose and 0.8% agar. Seeds were stratified for at least two days at 4°C and germinated aseptically at 25°C in a standard growth chamber (MLR-350, Sanyo Electric Co., Ltd.) with a 12 h/12 h light/dark cycle. 

P-MEXP-9941

Arabidopsis total RNA was isolated with the Plant RNeasy Kit (Qiagen, Chatsworth, CA), according to the manufacturer’s instructions.

P-MEXP-9942

Seven-day-old seedlings were irradiated at midday in a UV-B light field consisting of six Philips TL 40W/12 broadband UV fluorescent tubes (ƒÜmax = 310 nm, half-bandwidth = 40 nm, fluence rate = 7 W/m2) filtered through 3-mm transmission cut-off filters of the WG series with half-maximal transmission at the indicated wavelength (WG305 and WG327; Schott, Germany). Spectral energy distributions of UV-B sources were measured with an OL 754 UV-visible spectroradiometer (Optronic Laboratories, Orlando, FL), and are described in Ulm et al. 2004. After 15 minutes irradiation of the seedlings, plates were immediately returned to the standard growth chamber for 45 minutes. 

P-MEXP-9943

Whole plant tissue of at least 50 Arabidopsis seedlings was harvested, snap-frozen in liquid nitrogen and stored at -80°C until further processing.

P-MEXP-9944

Ten-µg aliquot of total RNA was reverse-transcribed using the Affymetrix cDNA synthesis kit as per manufacturer’s instructions (Affymetrix, Santa Clara, CA). The oligonucleotide used for priming was 5’-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3’ (Genset Oligo, France) as recommended by Affymetrix. Double-stranded cDNA was purified by phenol:chloroform extraction and the aqueous phase was removed by centrifugation through Phase-lock Gel (Eppendorf). The cDNA was then in vitro transcribed in the presence of biotinylated UTP to give labelled cRNA using the Affymetrix IVT kit. The cRNA was purified using RNAeasy clean-up columns (Qiagen). To improve the recovery from the columns, the elution water was spun into the matrix at 27 g and then left to stand for one minute prior to the standard 8000 g centrifugation recommended by Qiagen. The cRNA was fragmented by heating in fragmentation buffer (40mM Tris-acetate pH8.1, 100mM KOAc, 30mM MgOAc) as recommended by Affymetrix. The Affymetrix eukaryotic hybridization controls were added to the sample prior to hybridization as per manufacturer’s instructions.

Transcription profiling of Arabidopsis wild type, cop1-4, hy5-1 mutant seedlings exposed to polychromatic radiation

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