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Sample GSM7432877

Query DataSets for GSM7432877

Status

Public on Nov 30, 2023

Title

P-S6 control replicate 3

Sample type

SRA

Source name

Root tissue (18 days-old plants)

Organism

Gossypium barbadense

Characteristics

treatment: Control (ctl)
genotype: Pima-S6

Treatment protocol

Two treatments control (ctl) and FOV4 infected (FOV4) plants. To produce inoculum, the isolate was grown in 9-cm-diameter Petri dishes containing 20 ml potato dextrose agar (PDA) with 3 mM streptomycin per liter at room temperature for 1 week. Then a few patches of 2-3-mm-squre of fresh grown culture were cut and put into 500 ml flasks containing potato dextrose liquid medium with 3 mM streptomycin at 28 oC with rotation for 3-4 days. The conidial suspension was then filtered through eight layers of cheesecloth to remove hyphae, quantified with the aid of a hemacytometer, and diluted with water to obtain 1 x 106 conidia per ml. Cultivars or genotypes were germinated in seedling trays for one week and then seedlings were uprooted. Roots were rinsed with tap water and immediately dipped for 3 min into an aqueous suspension of FOV4 inoculum containing 1 x 106 conidia per ml. The non-inoculated control plants were processed the same way except they were dipped into water without FOV4. Seedlings were then transplanted into steam-sterilized U.C. Mix #2 (Baker, 1957) soil in new pots with five plants in each pot and placed in a warm greenhouse maintained between 24-28 degrees Celcius. At 11 days after inoculation (dai), the plant and soil ball were removed from the pots, and the soil removed gently from roots by washing under continuous water flow. The washed roots were placed immediately in 50ml screw-top plastic vials, liquid nitrogen was added, and the vials placed in a -80 C freezer with the tops off to allow vaporization of the liquid N.

Growth protocol

Cotton plants were grown in a Greenhouse in pots with soil. Air temperatures in the greenhouse were maintained between 280 C and 350 C during the day and 240 C at night.

Extracted molecule

total RNA

Extraction protocol

The root samples from each genotype were grounded in liquid nitrogen, and total RNA was isolated three biological replicates following the manufacturer’s instructions using Spectrum TM Plant Total RNA kit (Sigma-Aldrich, St. Louis, MO, USA). The yield and purity of RNA were analyzed with a ND-1000 Spectrophotometer (Nano Drop Technology, Wilmington, DE, USA). Only RNA samples with 1.8 - 2.2 ratio of absorbance 260/280 nm were used for analysis.
For each genotype, 2 mg of RNA from each of the three biological-replicates used for cDNA library preparation. The cDNA libraries were prepared following the TruSeq RNA Sample Preparation v2 low sample (LS) protocol guide (Illumina Inc., San Diego, CA USA)

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

FOV4 resistant genotype

Data processing

We first performed quality assessment of the resulting reads from the Illumina platform using FastQC (version 0.11.9; https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and processed sequencing libraries using Trimmomatic (version 0.39;) to remove adapter read sequences.
We then quantified gene expression using the pseudo-alignment RNA-seq quantification program kallisto version 0.46.1.
We then integrated transcript-level abundances from kallisto tocount-based statistical analysis in edgeR (version 3.13) to produce the table of read counts per locus.
Assembly: TheG. barbadense P-S6 genome release was used as a reference genome to align the reads(https://doi.org/10.1186/s12864-022-09102-6)
Supplementary files format and content: The table of counts is a tab-separated plaint text file (.txt) which contains the counts per million reads corresponding to each locus per library (rows), and each RNA-seq library sample (columns) is named according to genotype, condition and biological replicate (for example SJ2_ctl1 would be Acala SJ2-control conditions-replicate 1; rkn means root-knot nematode infested).

Submission date

May 30, 2023

Last update date

Nov 30, 2023

Contact name

Mauricio Ulloa

E-mail(s)

Mauricio.Ulloa@usda.gov

Phone

8063681659

Organization name

USDA-ARS