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Sample GSM5050310

Query DataSets for GSM5050310

Status

Public on Jan 30, 2021

Title

C_3_S28_L001_R1_001

Sample type

SRA

Source name

Stem and leaf tissue

Organism

Solanum lycopersicum

Characteristics

tissue: Stem and leaf tissue
treatment: untreated
cultivar: Moneymaker

Treatment protocol

Psyllid infestation were initiated when plants were six weeks old. Leaves branching below the apical meristem (i.e., leaves like the ones sampled for the transcriptome analysis) were caged with a small, white organza bag (amazon.com). Restricting psyllids to these leaves exposed them to systemic response of the plant to any prior infestation. Each bag either had no psyllids (control plants) or three adult male psyllids (psyllid-infested plants). Males were chosen to avoid the potentially confounding effect of oviposition on tomato gene expression. Seven days after infestation, caged tomato leaves were removed with a bleach-sterilized razor blade. Three weeks later, the top-most, fully developed leaf was sampled from each plant and immediately flash-frozen in liquid nitrogen. Samples were transferred to Eppendorf tubes and kept submerged under liquid nitrogen while ground with plastic, RNase-free pestles.

Growth protocol

Grown from seed in Metro-Mix 900 (Sun Gro Horticulture, Agawam, MA) soil and individually transplanted to 10 x 10 cm square pots four weeks later. Plants were watered every other day and fertilized weekly according to the manufacturer’s recommendation (Miracle-Gro® Water Soluble Tomato Plant Food; 18-18-21 NPK). All experiments were conducted at the same photoperiod (16: 8) and temperature (22±2°C).

Extracted molecule

total RNA

Extraction protocol

Total RNA extraction was performed on leaf tissue harvested three weeks after psyllid infestation using the Plant RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. Three biological replicates were sequenced per treatment (i.e., uninfested and psyllid-infested, six samples total). One fully-develop leaf and petiole were removed per biological replicate using sterilized razor blades. The top-most leaf was sampled to ensure that the gene expression changes observed were more likely to be associated with a plant systemic response. Samples were ground using sterilized plastic pestles. RNA samples were treated with RNase-Free DNase (Qiagen). Any remaining DNA was removed using the TURBO DNA-free™ Kit (Life Technologies, Carlsbad, CA). All remaining RNA was stored at -80°C for downstream quantitative reverse transcription PCR (RT-qPCR) validation. The isolated RNA was submitted to the Texas A&M Genomics and Bioinformatic Service for quality analysis, library preparation, and sequencing.
cDNA libraries were developed using the TruSeq RNA Library Prep Kit v2 (Illumina ®; San Diego, CA) following the manufacturer’s protocol, generating 2 Х 150 bp read lengths

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Sequence cluster identification, quality prefiltering, base calling, and uncertainty assessment were done in real time using Illumina’s HCS 2.2.38 and RTA 1.18.61 software with default parameter settings.
Alignment using HISAT2-index-align2.1 on the CyVerse platform mapping to the S. lycopersicum genome (vSL3.0)
Assembly using StringTie-1.3.3 on the CyVerse platform mapping hits to known transcripts based on the S. lycopersicum genome vITAG3.2 annotation and made non-redundant with StringTie-Merge
Ballgown analysis of FPKM values for each Sample using R
Genome_build: Solanum lycopersicum genome (vSL3.0)
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

Submission date

Jan 29, 2021

Last update date

Jan 31, 2021

Contact name

Kyle Harrison

E-mail(s)

kyle.harrison@usda.gov

Organization name

USDA-ARS

Department

Entomology