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Sample GSM3852664

Query DataSets for GSM3852664

Status

Public on Jun 05, 2019

Title

Barley-Shoot-SALT3

Sample type

SRA

Source name

Barley-Shoot-SALT3

Organism

Hordeum vulgare

Characteristics

developmental stage: seedling stage
passages: 6-7
accession: XZ26
tissue: Shoot
treatment: SALT (100 mM NaCl)

Treatment protocol

Salt treatment was initiated to barley and rice plants by increasing 50 mM NaCl a day to reach a final concentration at 100 mM in the hydroponics. The solution without NaCl addition was used as the control.

Growth protocol

Seeds of Tibetan wild barley accession XZ26 and Japonica rice cultivar Nipponbare were disinfected for 30 min with 2% H2O2, rinsed 5 times using distilled water and transferred onto moist filter paper in a germination box, then placed in a growth chamber (22/18 °C, day/night) in dark. After germination, the uniform seedlings of barley and rice were transplanted into 15 L black plastic containers filled with hydroponic solution as described by Fu et al (2018). All seedlings were grown in a controlled growth room at 26 °C of 14 h day/20 °C of 10 h night, with fluorescent lamps at 250 μmol m−2 s−1.

Extracted molecule

total RNA

Extraction protocol

After 7 d of salt treatments, fresh roots and the latest completely expanded leaves of barley (XZ26) and rice (Nipponbare) were sampled from both salt treatment and control, and immediately stored in liquid nitrogen for further transcriptomic analysis. Three biological replicates were done. Total RNA was extracted from approximately 0.1 g fresh tissues using the TRIzol Reagent (Invitrogen, Carlsbad, CA), and then was purified by the RNeasy Mini Kit (Qiagen, Germantown, MD). The concentration of total RNA was determined by Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, CA).
The RNA library construction, sequencing and data processing were performed as described by Wang et al (2016). Briefly, mRNA enrichment was obtained from total RNA by poly-T oligo-attached magnetic beads and randomly fragmented into small pieces. Double-stranded cDNA was synthesized using mRNA fragments with random hexamer-primers. The cDNA fragments were amplified and enriched by PCR and purified with adapters ligated at both ends using Illumina TruSeqTM RNA Sample Preparation Kit (Illumina, San Diego, CA). Then, PCR products were loaded onto Illumina HiSeq2500 platform (Illumina, San Diego, CA) for 2×100 bp pair-ends sequencing. Raw reads were trimmed by removing adaptor sequences, empty reads and low quality reads (Q<30 and length<50 bp) to get clean data. Sequence annotation was performed using Tophat (http://ccb.jhu.edu/software/tophat/index.shtml) based on the released reference genome of barley cv. Morex and rice cv. Nipponbare from Ensembl Genomes 2013 (http://plants.ensembl.org/index.html). For gene expression analysis, transcript abundance of each gene was normalized by the fragments per kilo-base per million fragments mapped reads (FPKM) method (Li et al., 2018). The ratio of [FPKM (treatment)]/[FPKM (control)] was calculated as the change fold for each gene. Differentially expressed genes (DEGs) were identified by following criterion: fold>2 and false discovery rate (FDR) <0.001. GO annotation and KEGG analysis were done using Blast2GO software version 3.2 (https://www.blast2go.com) based on the sequences of DEGs.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

S12

Data processing

The cDNA fragments were amplified and enriched by PCR and purified with adapters ligated at both ends using Illumina TruSeqTM RNA Sample Preparation Kit (Illumina, San Diego, CA).
PCR products were loaded onto Illumina HiSeq2500 platform (Illumina, San Diego, CA) for 2×100 bp pair-ends sequencing.
Raw reads were trimmed by removing adaptor sequences, empty reads and low quality reads (Q<30 and length<50 bp) to get clean data.
Sequence annotation was performed using Tophat (http://ccb.jhu.edu/software/tophat/index.shtml) based on the released reference genome of barley cv. Morex and rice cv. Nipponbare from Ensembl Genomes 2013 (http://plants.ensembl.org/index.html).
Transcript abundance of each gene was normalized by the fragments per kilo-base per million fragments mapped reads (FPKM) method,The ratio of [FPKM (treatment)]/[FPKM (control)] was calculated as the change fold for each gene. Differentially expressed genes (DEGs) were identified by following criterion: fold>2 and false discovery rate (FDR) <0.001. GO annotation and KEGG analysis were done using Blast2GO software version 3.2 (https://www.blast2go.com) based on the sequences of DEGs.
Genome_build: reference genome of barley cv. Morex and rice cv. Nipponbare from Ensembl Genomes 2013 (http://plants.ensembl.org/index.html)
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

Submission date

Jun 04, 2019

Last update date

Jun 05, 2019

Contact name

Liangbo Fu

Organization name

Zhejiang University

Department

college of agriculture and biotechnology

Lab

Crop science A463