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Sample GSM4613108

Query DataSets for GSM4613108

Status

Public on Jun 13, 2020

Title

Arcanum_T8h_P3

Sample type

SRA

Source name

Leaves

Organism

Solanum arcanum

Characteristics

cultivar: LA2157
tissue: leaves
hpi: 8
treatment: Infection with pathogenic strain Cmm 156

Treatment protocol

Forty days after germination of both plant tomato species were infected with the pathogenic strain Cmm 1569. The bacterial cells were propagated in 802 medium culture (2g/L yeast extract, 1g/L polypeptone and 0.92 g/L magnesium sulfate). For inoculation, three biological replicates per each tomato species (S. lycopersicum and S. arcanum) were inoculated in the first proximal internode above ground of each plant with an insulin syringe with a concentration of 5x10⁷ CFU (OD = 0.2). After inoculation, tomato plants were sampled (true leaves next to inoculation site) at 0, 8 and 24 h post inoculation. Biological samples were immediately frozen in liquid nitrogen and stored at -80°C.

Growth protocol

All experiments were carried out in controlled conditions, as described below, and plant residues were sterilized. Before germination, all tomato seeds were sterilized in a 10% extran solution for 15 min. Subsequently, three washes with deionized water and one wash with 70% ethanol solution were performed, followed by a 10% bleach solution wash for 10 min and rinsing three times in deionized water. Tomato seeds were germinated in a commercial mix substrate (Sunshine Mix #3, Sun Grow Horticulture, Vancouver, BC, CA) in a bioclimatic chamber at 25°C and 16/8 h light and dark period. Two weeks later, plants were transferred to greenhouse conditions with a controlled temperature max 28°C, min 20°C and humidity of 55-60%

Extracted molecule

total RNA

Extraction protocol

The 18 samples (nine each of S. lycopersicum and S. arcanum) were processed and sequenced by BGI Hong Kong (Beijing Genome Institute, https://www.bgi.com/global/) as follows. RNA quality of the samples (OD 260/280 & 260/230) was tested by NanoDropTM after total RNA extraction. Total RNA samples concentration, RIN, 28S/18S, was determined by using the Agilent 2100 Bioanalyzer (https://www.agilent.com/). According to the TruSeq RNA Sample Prep Kit v2 (Illumina), mRNA molecules were purified using poly-T oligo attached magnetic beads. Following purification, for each sample mRNA was fragmented into small pieces using divalent cations at 94°C. The purified RNA was used as template for cDNA synthesis.
RNA libraries prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

Methodology 01, Mapping to the ITAG3.2 genome: Sequence reads were mapped to ITAG3.2 genome using Hisat 2 with parameters --non-deterministic; quantifications to each exon per gene was performed with FeatureCounts from SubRead package witch parameters -p -T $n -J -M --fraction --largestOverlap --fracOverlap 0.75.
Methodology 02, Mapping to the semi-de-novo transcriptome: Sequence reads were mapped and quantified to the semi-de-novo transcriptome (ITAG3.2 transcriptomes plus a Trinity transcritome with thos sequence reads un-mapped to ITAG3.2 genome) using Kallisto with default parameters.
Methodology 01, Mapping to the de-novo transcriptome: Sequence reads were mapped and quantified to the de-novo transcriptome (generated with Trinity using all sequence reads) using kallisto with default parameters.
Normalized coutns were obtained with the cpm() function from edgeR R package.
Genome_build: ITAG3.2, Sollanum Lycopersicum
Supplementary_files_format_and_content: Matrix tables with raw transcript counts and cpms for every sample in each methdology: mapping to ITAG3.2 genome, mapping to semi-de-novo transcriptome, and mapping to de-novo transcriptome.

Submission date

Jun 12, 2020

Last update date

Jun 13, 2020

Contact name

Cesaré Ovando-Vázquez

E-mail(s)

cesare.ovando@ipicyt.edu.mx

Organization name

CONACyT IPICyT

Department

CNS