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Sample GSM3106678

Query DataSets for GSM3106678

Status

Public on Apr 20, 2019

Title

Yy97 tm2

Sample type

SRA

Source name

Yanyan97

Organism

Nicotiana tabacum

Characteristics

cultivar: Yanyan97
tissue: stem
developmental stage: 5-6 leaf stage
infection: Ralstonia solanacearum

Treatment protocol

Pathogen inoculation was performed at 4 to 5 true leaves. Virulence R. solanacearum strain CQPs-1 (kindly supplied by Pro. Wei Ding, College of Plant Protection, Southwest University) was used. This pathogen is belongs to phylotype I (Liu et al. 2017). The bacterium was reproduced in B medium (Boucher et al. 1985) at 28 ±1°C, 180 rpm in a ZHWY-2102C shaking incubator (Shanghai ZHICHENG Analytical Instrument Manufacturing CO., LTD, Shanghai, China) for 24 hours. The bacterial suspension was adjusted to an OD600 of 0.1 with sterile deinonized water. For pathogen inoculation, to each plant, diluted pathogen suspension of 20 ml was poured to the stem base. For control plants, equal volume of sterile water was added with the same method. There were at least 24 plants for each treatment. After inoculation, both the control and inoculated plants were grown at same condition as described above with except temperature was adjusted to 28 ± 1°C.

Growth protocol

Resistant (R line, Yanyan 97,Yy97) and susceptible (S line, Honghuadajinyuan，Hd) tobacco cultivars were used in this study. For each germplasm, ca. 100 seeds were surface-sterilized by immersion in 2% sodium hypochlorite (NaClO) for 5 min, followed by three rinses in sterile water. Subsequently, the seeds were plated in Petri dishes (9 cm in diameter) on Whatman No. 3 filter paper moistened with sterile water. Then the seeds were cultivated at 28±1ºC in dark in a chamber. After germination, the plantlet were individually transplanted into plastic pot (9 cm in diameter and 10 cm in depth) filled with pasteurized nutritious soils (clay: sand: organic matter = 3:1:1, v/v). The seedlings were grown at 25 ± 1°C with a 12 h photoperiod，80% ± 1 RH , under light intensity of 5000 lux, in a greenhouse of Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science (IBFC, CAAS).Resistant (R line, Yanyan 97,Yy97) and susceptible (S line, Honghuadajinyuan，Hd) tobacco cultivars were used in this study (Ji et al., 2000). For each germplasm, ca. 100 seeds were surface-sterilized by immersion in 2% sodium hypochlorite (NaClO) for 5 min, followed by three rinses in sterile water. Subsequently, the seeds were plated in Petri dishes (9 cm in diameter) on Whatman No. 3 filter paper moistened with sterile water. Then the seeds were cultivated at 28±1ºC in dark in a chamber. After germination, the plantlet were individually transplanted into plastic pot (9 cm in diameter and 10 cm in depth) filled with pasteurized nutritious soils (clay: sand: organic matter = 3:1:1, v/v). The seedlings were grown at 25 ± 1°C with a 12 h photoperiod，80% ± 1 RH , under light intensity of 5000 lux, in a greenhouse of Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science (IBFC, CAAS).

Extracted molecule

total RNA

Extraction protocol

To prepare samples for Illumina sequencing, total RNA of each sample was extracted using an EASYspin plus Total RNA kit (Aidlab, Beijing, China) following the protocol. Quality and yield of RNA were determined by agarose gel electrophoresis and Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), respectively.
Total RNA was extracted from the stem samples with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and purified with Plant RNA Purification regent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. The concentration and purity of total RNA was determined using NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was examined by Agilent2100 bioanalyzer (Agilent, Santa Clara, CA, USA).Illumina sequencing was performed at Majorbio Bio-pharm Technology Co. Ltd, Shanghai, China (http://www.majorbio.com/). For transcriptome sequencing, eight individual libraries (Yy97ck1, Yy97ck2, Yy97tm1, Yy97tm2, Hdck1, Hdck2, Hdtm1 and Hdtm2) were constructed using 5 μg of total RNA. Briefly, poly (A) mRNA was extracted from the total RNA sample using Oligo (dT) magnetic beads. mRNA was fragmented into 200–500 nt pieces by adding a fragmentation buffer. First-strand cDNA was synthesized using SuperScript II reverse transcriptase (Life Technologies, Inc.) and random hexamer primers. After generation of second-strand cDNA, the double-strand cDNA was end-repaired, and a single ‘A’ base and indexed adapters were ligated to the fragments. The cDNA library was constructed with the Illumina Paired End Sample Prep kit (Illumina, USA), quantified by TBS380 (Picogreen, Invitrogen, USA) and was then sequenced on the Illumina HiSeqTM2500 (2×150 bp read length) platform.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

yy97_geneExpression.txt

Data processing

RNA-Seq: the cDNA library with a fragment length range of 300 bp (±25 bp) was constructed and the paired-end sequencing was performed by using the Illumina sequencing platform (HiSeqTM 2500).
Filtering: Adapter sequences were removed, higher N rate sequences, and low-quality reads (less than 13 bp or reads with unknown nucleotides larger than 5%) were filtered with the SeqPrep and Sickle software.
Gene annotation: High-quality reads were mapped to the tobacco (cultivar K326) genome (https://www.ncbi.nlm.nih.gov/assembly/GCA_000715075.1/) using Hisat2 (http://ccb.jhu.edu/software/hisat2/index.shtml) and annotated based on the tobacco genome sequence. The mapped reads were then assembled using Stringtie v1.3.3b (http://ccb.jhu.edu/software/stringtie/) with default parameters, and gffcompare utility was used to determine how many assembled transcripts match annotated genes and how many are novel transcripts. Functional annotation of new transcripts was performed through a match search against SWISS-Prot and GenBank-NR database using DIAMOND (e-value of 1e-5).
Gene Expression Analysis: Gene expression levels were measured in the RNA-Seq analysis as fragments per kilobase of exon model per million mapped reads (FPKM). The DEseq software was used to identify differentially expressed genes in pair-wise comparison, and the results of all statistical tests were corrected for multiple testing with the Benjamini–Hochberg false discovery rate (FDR < 0.01).
Supplementary_files_format_and_content: The .txt files contain the gene name, fpkm value, the number of total reads and the number of unique reads

Submission date

Apr 20, 2018

Last update date

Apr 20, 2019

Contact name

Yongting Yu

Organization name

Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science

Street address

No 348, Xianjiahu West Rd.

City

Changsha