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Sample GSM4368184

Query DataSets for GSM4368184

Status

Public on May 25, 2020

Title

Rosettes, Mannitol_1

Sample type

SRA

Source name

Rosettes

Organism

Arabidopsis thaliana

Characteristics

stress treatment: Mannitol
tissue: Rosettes
biological replicate: Rep1

Treatment protocol

For salt stress treatment, plants were watered with 150 mM NaCl solution 16 h before sampling. The osmotic stress treatment was applied by watering plants with 200 mM mannitol 16 h before sampling. For heat treatment, plant trays were transferred to a growth cabinet pre-adjusted at 35°C for 4 hours before sampling. To avoid the circadian clock effects that could interfere with the interactions between our treatments, we started the heat treatment after 3 h after light onset, and collected all samples at the midday where the plant metabolism is at a steady state. Double treatment of salt and mannitol was applied by watering plants with a mixed solution containing final concentration of 150 mM NaCl and 200 mM mannitol. For double treatments of heat with NaCl or mannitol, a fraction of the plants that were watered with NaCl or mannitol were moved after 12 hours to a growth cabinet pre-adjusted at 35°C for extra 4 h, making a total of 16 h before sampling. For multiple stress treatments, a patch of the plants treated with the double treatment of NaCl 150 mM and 200 mM mannitol were transferred after 12 h to a growth cabinet pre-adjusted at 35°C for extra 4 hours, making total of 16 h before sampling (Figure 1A). To apply multiple stress treatment similar to field conditions as much as possible, Arabidopsis plants were treated with salt and mannitol 1 hour before the light is off, and kept for 12 h before starting heat treatment. After 3 h of light turning on, plant trays were transferred to 35°C growth cabinet for extra 4 h making a total of 16 h treatment.

Growth protocol

Seeds of wild-type Arabidopsis thaliana ecotype Columbia-0 (Col-0; WT) were sown on soil. After stratification at 4°C for 2 days, the seeds were transferred to a long-day (16 h light/8 h dark) growth chamber using a mix of bulbs from Spectralux®Plus NL 36W/840 (Radium) and Fluora L 36W/77 (Osram) with a light intensity of approximately 100 µE m-2 s-1 and a temperature regime of 22°C day/18°C night temperatures. Two-week old seedlings were transplanted to new soil. After another 14 days of growth, plants were subjected to the stress treatments.

Extracted molecule

total RNA

Extraction protocol

Rosettes were harvested and immediately frozen in liquid nitrogen. Tissue was ground into fine powder using liquid nitrogen, and then kept at -80°C until use. Total RNA was extracted using the Promega Kit (SV Total RNA Isolation System, spin protocol). The integrity of the extracted RNA was anaylsed by gel electrophoresis and its concentration was measured using a NanoDrop (ND-1000) spectrophotometer.
Libraries were prepared using the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, United States) and quantified with a Qubit 2.0 (Invitrogen, Waltham, MA, United States). Samples were multiplexed with 12 libraries per lane and sequenced in paired-end mode (Rapid Run, 100 bp read length) on an Illumina HiSeq 2500 platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

M_1

Data processing

Illumina Casava-1.9software used for basecalling.
After successful quality control with the Fast QC software (v0.11.5), Illumina reads were quantified by mapping against the Arabidopsis reference transcriptome (Araport 11 representative CDS, Araport11_genes.201606.cds.fasta.gz, retrieved from www.araport.org) using Kallisto v 0.45.1 (Bray et al., 2016) in default mode with 50 times bootstrapping (option -b 50) for sleuth. Kallisto provides normalized transcript abundance as transcripts per million (tpm).
Differential transcript abundance comparing each of the treatments against the control were determined using the likelihood ratio test implemented in sleuth v0.30.0 (Pimentel et al., 2016). An alpha of 0.05 was chosen after adjusting p-values (thereafter termed ‘q-value’) for multiple hypothesis testing via Benjamini–Hochberg correction (Benjamini and Hochberg, 1995) as implemented in sleuth. The ‘b value’ supplied by sleuth was used as adapted log2-fold change (LFC) between the respective treatment and control.
Means and standard deviation of tpm of biological triplicates group were calculated.
Genome_build: Araport 11 representative CDS, Araport11_genes.201606.cds.fasta.gz, retrieved from www.araport.org
Supplementary_files_format_and_content: Excel file including Arabidopsis locus ID, brief annotation, mapman annotation, raw estimated counts (kallisto), tpm (kallisto) for each sample and mean and standard deviation of tpm for biological triplicates, sleuth results of differential gene expression comparing each treatment against control.

Submission date

Mar 02, 2020

Last update date

May 25, 2020

Contact name

Andreas PM Weber

E-mail(s)

aweber@hhu.de

Organization name

Heinrich-Heine-University

Department

Institute for Plant Biochemistry