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Sample GSM3273401

Query DataSets for GSM3273401

Status

Public on May 29, 2019

Title

uvr8-2-blue0-R2

Sample type

SRA

Source name

whole rosette

Organism

Arabidopsis thaliana

Characteristics

genotype: uvr8-2
developmental stage: 3 weeks old plants
solar treatment: Blue0
tissue: whole rosette

Treatment protocol

Outdoors, five filtered sunlight treatments (UVA+B, UVA350, UVA, UV0 and blue0) were created by using different plastic sheets and films as wavelength-selective long-pass optical filters. These treatments were randomly assigned within four blocks or biological replicates. Plants were moved outdoors under filters on 21.08.2014 at sunrise (07:30 to 08:15). Samples from the different filter frames were collected for RNA-seq analysis after 6h of radiation treatment, at solar noon (13:30 to 14:15). The samples were harvested sequentially by block with treatments and genotypes in random order within each block.

Growth protocol

Seeds of Arabidopsis wild-type Ler and the photoreceptor mutants uvr8-2 (Brown et al., 2005) and cry1cry2 (Neff and Chory, 1998) were sown in plastic pots (8 × 8 cm) containing a 1:1 mixture of peat and vermiculite and kept in darkness at 4°C for 3 days. Thereafter the pots were transferred to environmentally controlled growth rooms at 23°C/19°C (day/night) and 70%/90% relative humidity under 12 h photoperiod with 280 µmol m−2 s−1 PAR irradiance from white fluorescent lamps. One seedling was transplanted into a new plastic pot (5 × 5 cm) using the same substrate type. After transplanting, plants were kept for 14 days in growth rooms. Rosco filter E-color 226 was used to avoid plants receiving the small amount of UV-A radiation emitted by lamps. Thereafter, five plants/pots from each genotype were randomly assigned to individual plastic trays which were moved outdoors to the filtered sunlight treatments.

Extracted molecule

total RNA

Extraction protocol

Each biological sample collected consisted of five pooled rosettes frozen in liquid nitrogen and stored at –80°C. The rosette leaves from each pooled sample were ground with mortar and pestle in liquid nitrogen. An aliquote from the powdered sample was separated for gene expression (RNA-seq and qPCR). RNA was extracted with with GeneJet Plant RNA purification Mini Kit (Thermo Scientific). RNA quality and concentration were measured using Agilent 2100 Bioanalyzer and NanoDrop.
RNA-seq library preparation and sequencing was performed at the Institute of Biotechnology, University of Helsinki using two pooled biological replicates. Each replicate consisted of RNA from two different experimental blocks in the field. Libraries were prepared using 1µg total RNA following instructions from Illumina TruSeq Stranded mRNA Library Prep Kit.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

The quality of raw reads was first inspected in Chipster using FastQC.
Removal of adapter sequences and trimming and cropping of the reads was done using Trimmomatic-0.33 (Bolgert et al., 2014) in single-end mode. The bases with a quality score less than 20 were trimmed from the beginning and the end of the sequences, and the reads shorter than 30 bases were removed from the analysis (-phred33, LEADING:20, TRAILING:20 and MINLEN:30).
Filtered reads were mapped to the Arabidopsis transcript reference database AtRTD2 (Zhang et al., 2017) using Kallisto V-0.43.0 (CMD:quant) (Bary et al., 2016) with 4000 bootstrap sets. The final count table for each biological replicate was obtained as the mean of the bootstrap runs.
Genome_build: Arabidopsis transcript reference database AtRTD2
Supplementary_files_format_and_content: Matrix-table including gene counts for each sample

Submission date

Jul 17, 2018

Last update date

May 29, 2019

Contact name

Luis Morales

E-mail(s)

luis.morales@oru.se

Phone

+46 19 301033

Organization name

Örebro University