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Sample GSM2358973

Query DataSets for GSM2358973

Status

Public on Mar 07, 2017

Title

T25_1

Sample type

SRA

Source name

T25 leaves

Organism

Vitis vinifera

Characteristics

tissue: leaf
induction stage: T25

Treatment protocol

When the sixth leaves (from base to apex) of grapevines became mature, all grapevines were divided into four groups and acclimated for two days in a controlled environment room (70% average relative humidity, 25/18 (12 h/12 h) day/night cycle and PAR at 800 μmol m−2 s−1). On day 3, the grapevines were subjected to the following treatments: (1) the plants of the control group were maintained at the optimal day/night temperature (25°C/18°C) in the above growth room; (2) the plants of the treatment groups were exposed to 35°C, 40°C or 45°C from 11:30 to 13:30 (the conditions were the same as the control, except for temperature). The fourth to sixth leaves (from base to apex) of each plant were detached at 13:30 (the end of the heat stress treatment). Each biological replicate included three plants, and three replicates were used for both the three treatments and the control. Leaves were frozen in liquid nitrogen immediately and stored at −80°C for further analysis.

Growth protocol

One-year old ‘Jingxiangyu’ (Vitis vinifera L.) grapevines were planted in pots, then grown in a greenhouse at 70-80% relative humidity at 18-25°C, with the maximum photosynthetically active radiation (PAR) at approximately 1,000 μmol photons m−2 s−1.

Extracted molecule

total RNA

Extraction protocol

Total RNAs from leaves were extracted using Trizol (Invitrogen, 15596-018) according to the manufacturer’s instructions. Highly purified and intact mRNAs were enriched from total RNAs using Dynabeads® mRNA purification kit (Ambion, 61006). RNAs were fragmented into ~ 300 nt fragments by 1 min incubation at 94℃ in fragmentation buffer (10 mM ZnCl2, 10 mM Tris-HCl, pH 7.0). The fragmentation reaction was stopped with 50 mM EDTA, followed by standard ethanol precipitation and collected for sequencing and expression validation.
For the strand-specific RNA-seq, fragmented RNAs were re-suspended in H2O and used for library generation with mRNA sequencing kit (Illumina Inc. San Diego, CA). Sequencing was carried out on Illumina HiSeq 4000 sequencer (Illumina Inc. San Diego, CA) according to the manufacturer’s instructions and 150 nt paired-end sequencing reads were generated.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Treated at 25 ℃

Data processing

Base-calling were performed using RTA and CASAVA.
The quality of sequencing data was estimated using FastQC (v0.11.3). Then adaptors and low quality bases were removed using Trimmomatic (v0.33).
Clean reads from all samples were pooled together and feeded to de novo RNA-seq assembly software Trinity (v2.0.6) with default parameters.
The expression of assembled transcripts and genes were quantified using RSEM (v1.2.19).
Genome_build: vitis28
Supplementary_files_format_and_content: identified alternative splicing events + counts

Submission date

Oct 24, 2016

Last update date

May 15, 2019

Contact name

Lijun Wang

E-mail(s)

[email protected]

Organization name

IBCAS

Street address

No.20 Nanxincun, Xiangshan, Beijing 100093, China