Comment[GEAAccession]	E-GEAD-947
MAGE-TAB Version	1.1
Investigation Title	Genome-wide analyses of early translational responses to elevated temperature and high salinity in Arabidopsis thaliana (CBX69)
Experiment Description	We investigated genome-wide changes in mRNA translation in Arabidopsis thaliana suspension cell cultures exposed to brief perids of two types of stress: elevated temperature (37degree C) and high salinity (200 mM NaCl). To this end, we subjected polysomal RNA and non-polysomal RNA from sucrose gradient fractionated cell lysates to the co-hybridization on Agilent Arabidopsis 3 Oligo Microarrays. The ratio of signal intensities (polysomal RNA: non-polysomal RNA) was used as an indicator of the translation state for each transcript. To inspect coordination of changes in translational profiles with transcriptional profiles, we also isolated total RNAs from the same cells used for translational profiling experiments and investigated changes in accumulated transcript levels in response to each stress using the microarray. Two biological replicates were analyzed.
Experimental Design	translational bias design	stimulus or stress design
Experimental Factor Name	sample_name
Experimental Factor Type	sample_name
Person Last Name	Matsuura	Kato
Person First Name	Hideyuki	Ko
Person Affiliation	Nara Institute of Science and Technology (NAIST)	
Person Roles	submitter	
Public Release Date	2025-01-31
PubMed ID	20089509	11160997
Protocol Name	P-GEAD-3866	P-GEAD-3867	P-GEAD-3868	P-GEAD-3869	P-GEAD-3870	P-GEAD-3871	P-GEAD-3872	P-GEAD-3873
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol	growth protocol	treatment protocol
Protocol Description	none	Approximately 0.3 g of stress-treated cells or the corresponding control cells were pulverized in liquid nitrogen with a mortar and pestle. The resulting frozen powder was homogenized in 1.5 ml of buffer U (200 mM Tris-HCl, pH 8.5, 50 mM KCl, 25 mM MgCl2, 2 mM EGTA, 100 microg/ml heparin, 2% polyoxyethylene 10-tridecyl ether, and 1% sodium deoxycholate) and centrifuged at 15,000 x g for 10 min at 4degree C.  Aliquots of the supernatant were layered on 4.6 ml of 15-60% sucrose density gradient in buffer b (50 mM Tris-HCl, pH 8.5, 25 mM KCl, and 10 mM MgCl2) and centrifuged at 55,000 rpm for 50 min at 4degree C in a SW55 rotor (beckman Coulter, Fullerton, CA, USA).  The gradient was collected from the bottom using a peristaltic pump into 8 fractions of approximately 650 microl, with simultaneous recording of absorbance profiles at 254 nm using a UV monitor (Monitor UV-1; GE Healthcare, Little Chalfont, UK). Fraction 1 to 3 (polysome fraction) and 5 to 7 (nonpolysome fraction) were individually collected into tubes containing 8 M guanidine hydrochloride (final concentration, 5.5 M) and mixed. Direction of sedimentation is from fraction 8 to fraction 1. Concomitantly with the collection, known amounts of spike-in RNAs (Two-Color RNA Spike-In Kit; Agilent Technologies, Palo Alto, CA, USA) were added to both fractions as an external control. Equal volumes of Spike A and Spike b Mix were added to polysome and nonpolysome fractions, respectively. RNA was precipitated by addition of an equal volume of ethanol to the sucrose mixture, overnight incubation at -20degree C, and centrifugation at 10,000 rpm for 45 min in a JA-20 rotor (beckman Coulter). The resulting RNA precipitation was washed with 80% ethanol and purified using an RNeasy kit (Qiagen, Hilden, Germany) with on-column DNaseI treatment. RNA solution was further subjected to LiCl precipitation by the addition of LiCl at a final concentration of 1.5 M, followed by centrifugation. After ethanol precipitation, RNA pellet was resuspended in RNase-free water. RNA integrity was examined with an Agilent bioanalyzer 2100.  Approximately 0.1 g of cultured cells was harvested and pulverized as described above for the polysome fractionation assay. First, total RNA was isolated from the resulting frozen powder using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) followed by RNeasy mini spin columns (Qiagen) with oncolumn DNase I treatment. The reagents and kit were used according to the manufacturer's instructions. RNA integrity was examined with an Agilent bioanalyzer 2100 (Agilent Technologies).	Fluorescent cRNA was generated from polysomal and nonpolysomal RNA or total RNAs from stress-treated and control cells using a Low RNA Input Fluorescent Linear Amplifi cation Kit (Agilent Technologies). A 500 ng aliquot of RNA was subjected to reverse transcription using MMLV reverse transcriptase and an oligo(dT) primer containing the T7 promoter, and subsequently in vitro transcribed using T7 RNA polymerase, resulting in Cy3-labeled and Cy5-labeled cRNAs. The cRNA was purifi ed using RNeasy mini spin columns (Qiagen).	Polysomal RNAs and total RNAs derived from stress-treated cells were labeled with Cy3, and non-polysomal RNAs and total RNAs derived from the control cells were labeled with Cy5. Mixtures of 0.85 microg of Cy3-labeled and Cy5-labeled cRNAs (either polysomal and non-polysomal RNAs derived from anidentical cell lysate or total RNAs from stress-treated and corresponding control cells) were co-hybridized at 65  degrees C for 17 h on an Agilent Technologies 4 x 44K Arabidopsis 3 60mer oligo microarray.	Microarray slides were scanned using a Agilent Microarray Scanner at a pixel resolution size of 5 microm.	Total RNA (transcriptome) profiling data were normalized using a Linear and LOWESS method in Agilent Feature Extraction software. Polysomal RNA (translatome) profiling data were normalized via a Linear and LOWESS method followed by spike-in normalization utilizing a Two-Color RNA Spike-InKit (Agilent Technologies)., The fluorescent intensity of individual features (spots) on scanned images was quantified and corrected for background signal using Feature Extraction software (Agilent Technologies). To analyze only microarray data of high quality, only features that passed three criteria were used for further analysis. Features flagged in Feature Extraction software as non-uniform (IsFeatNonUnifOL and IsFeatPopnOL field), saturated (IsSaturated field) or low signal (IsWellAbovebG field) in either of the two biological replicates were omitted. The average of log10-transformed (Cy3/Cy5) values was calculated for genes mapped on more than two features on an array, and then for the two biological replicates., Fluorescent intensity of individual spots on scanned images was quantified and corrected for background signal using Agilent Feature Extraction software.	Aradidopsis thaliana suspension cell strain T87 was cultured in modifi ed LS medium with constant agitation at 120 r.p.m. at 22  degrees C under a 16 h light/8 h dark photoperiod. An aliquot of cell suspensions (4-8 ml) was transferred into 95 ml of fresh medium every week.	Aliquotes of three- or four-day-old suspension cultured cells from the same batch were subjected to 10 min of stress treatments (37?C or 200 mM NaCl at 22?C) or the corresponding control treatments (22?C or 0 mM NaCl at 22?C). After the treatments, cells were immediatly harvested by suction filtration, frozen in liquid nitrogen, and stored at -80?C. before harvesting, salt-stressed cells and the corresponding control cells were washed with 180 mM mannitol solution that was iso-osmotic relative to the culture medium.
SDRF File	E-GEAD-947.sdrf.txt
Comment[Number of channel]	single-channel
Comment[Array Design REF]	A-GEAD-152
Comment[AEExperimentType]	transcription profiling by array
Comment[SecondaryAccession]	CBX69
Comment[BioProject]	PRJDB20046
Comment[CIBEX Accept Date]	2010-01-01
Comment[CIBEX Public Release Date]	2010-02-15
Comment[Last Update Date]	2025-01-31