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Sample GSM4815463

Query DataSets for GSM4815463

Status

Public on Apr 01, 2021

Title

ESSEX HL 23 (HY5_HL_3)

Sample type

SRA

Source name

fully expanded leaf

Organism

Arabidopsis thaliana

Characteristics

genotype: hy5-2/Col-0
tissue: fully expanded leaf
treatment: 3.5 h high light

Treatment protocol

The HL exposure was a light intensity of 1100 (± 100) µmol m-2 s-1 from a white light emitting diode (LED) array (Isolight; Technologica Ltd, Colchester UK). Six plants, at 35 days post-germination, were exposed to HL for 3.5h. The same number of plants were selected to be LL controls. The HL exposure began 1h after subjective dawn and was carried out in the chamber the plants were grown in. At the end of HL exposure, one exposed plant, not destined to be sampled, was monitored for its response to HL by determining the degree of decline in the dark-adapted chlorophyll fluoerescence (CF) parameter Fv/Fm using a CF imager (Technologica Ltd., Colchester, UK) and compared with a LL control plant. This confirmed that the HL exposure and plants' responses proceeded as expected.

Growth protocol

Arabidopsis thaliana plants (Col-0; BBX32-10; hy5-2 ) were grown under a 8:16-h light:dark cycle at 23°C, 60% relative humidity, and light intensity of 150 µmol m-2 s-1. Individual Arabidopsis seed were sown onto a soil mix (Scotts Levingtons F2+S compost) in round pots (2.5 cm diameterx x 5cm) and placed for 3 d at 4°C for stratification before transferring to growth conditions for germination and growth . The Col-0 and hy5-2 genotypes were obtained from the Euorpean Arabidopsis Stock Centre, BBX32-10 seed was a kind gift from Dr Rajnish Khanna (then at Mendel Biotechnology Inc., Cailfornia, USA).

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from four individual plants (4 leaves per plant) per treatment (8 in total) using TRIzol reagent according to manufactuere's protocol (Invitrogen), followed by DNAase1 treatment, further phenol extraction, ethanol precipitation and re-suspension in sterile distilled water after brief drying. Concnetration of RNA was deterimined initially using a nanodrop spectrophotomer. Quality of the RNA and its concentration was checked on a Agilent 2100 Bioanalyzer using RNA 6000 nanochip according to the manufacter's protocol. The 24 samples (represneting 6 goups of biological replicates were confirmed by quantitative real time PCR as having no significant differences between them in the cDNA levels of transcripts of a PP2A reference housekeeping gene. Samples were delivered frozen on dry ice within 24h to the sequence service provider (Novogene, Cambridge).
This was conducted by Novogene (Cambridge, UK). The total RNA was enriched for mRNA using oligo-dT magnetic beads. After recovery of the enriched mRNA fraction, the RNA was fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamer primers and revrse transcriptase.After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The ds cDNA was purified from the reaction solution, end repaired and a 3' A added. Adaptors were then ligated onto the tailed cDNA fragments. Sequencing adaptors were a standard Illumina design. Adaptor - fragments were size selected (250-300 bp insert size) and enriched by PCR amplification. Librar concentration was initially quantified using a Qubit 2.0 fluorimeter (Life Technologies). Insert size was checked on an Agilent 2100 Bioanalyzer and quantified using quantitative PCR.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Sequence data was generated on a Illumina NovaSeq 6000 (model identifier HWI-ST1276; FlowCell ID- C1162ACXX). Raw unfiltered data deposited as FASTQ files (single-read). Raw sequence reads ranged from from 19861503 to 27395835. Sequence reads base calling using Illumina CASAVA software calculated from the Phred score to have an error rate of 0.03% in all reads and Q30 values of 92.83% to 94.2%.
Raw sequence data filtering to remove adapter sequences (5'Adapter (RA5) 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' ; 3' Adpter (RA3) 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC(6-nucleotide index)ATCTCGTATGCCGTCTTCTGCTTG-3 ; 6 nucleotide index sequence CGATGT), reads where >10% of base calls were uncertain and >50% of nucleotides in a sequence were of low qulaity (base quality call >20). Cleaned sequence reads ranged from 19826717 to 27063004.
Read alignment to reference genome (TAIR 10) conducted using the program HISAT2 v2.0.5 using the default parameters. >98% of filtered reads were mapped to the genome and of these 98.57% of aligned reads mapped to exons. Outputs are files are in BAM files for each sample.
Transcript abundance is the measure of gene expression and is expressed as Fragments Per Kilobase of transcript sequence per Million base pairs sequenced (FPKM) and is scored on reads that map to exons. The software used was HTSeq v0.6.1 in -m union mode.Within each treatment/genotype group of 4 the square of the Pearson correlation coefficient >0.92, with the exception of BBX32_HL_2 and BBX32_HL_4 comparison for which the value was 0.82.
Differential expression analysis between treatments used normalised mean values from each group (e.g. COL0, COL0_HL etc.) and was determined using the program DESeq2 v1.20.0 (normalisation method DEseq) to generate log2 fold change and p values determined using a negative binomial distribution estimation model. False discovery rate (Benajmini-Hochberg) generated padj. values ,which were regarded as significant at <0.05.
Genome_build: TAIR 10

Submission date

Oct 01, 2020

Last update date

Apr 01, 2021

Contact name

Ellie Jane Stallard

E-mail(s)

ellie.stallard@essex.ac.uk

Organization name

University of Essex

Department

School of Life Sciences