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Sample GSM8345882

Query DataSets for GSM8345882

Status

Public on Jun 25, 2024

Title

Root, 15-B20_S12

Sample type

SRA

Source name

root

Organism

Arabidopsis thaliana

Characteristics

tissue: root
genotype: B2
treatment: 0 NaCl

Treatment protocol

Concentrations of sodium chloride (NaCl). For root phenotype analyses and isoprene emission measurements, five-day-old seedlings were treated with 0, (mock), 50 (mild stress) or 100 (severe stress) mM NaCl for five days in solid MS medium. To study the primary root growth in soil rather than in an artificial substrate, NE (EV) and IE (B2 and C4) Arabidopsis plants were planted in homemade Rhizoboxes, special pots where root growth can be monitored. Plants were germinated and grown for two weeks in sterile Suremix soil (Michigan Grower Products Inc, Galesburg, MI, USA) and kept in the same controlled growth chambers used for plates.

Growth protocol

We used our previously developed A. thaliana ISPS-transgenic lines (Zuo et al., 2019). The full complementary ISPS DNA sequence including native transit peptide sequence from Eucalyptus globulus was placed under the Arabidopsis rubisco small subunit promoter rbcS-1A (At1g67090) to generate the transgenic, IE lines. After the ISPS sequence, an octopine synthase gene was placed as a transcriptional terminator of the transgene. The construct without EgISPS was also transformed with the vector to generate a NE EV control. After successful transformation, seven transgenic lines and three empty vector lines were characterized. Among the transgenic lines, B2 and C4 showed highest level of isoprene emission (Zuo et al., 2019). Thus, we selected B2 and C4 transgenic lines as IE, together with EV-B3 (referred as EV hereafter) as NE for the current study. The A. thaliana seeds were surface sterilized with 70% ethanol and 5% bleach solution for 5 and 10 min, respectively, followed by washing with sterilized milli-Q water for five times. Seeds were placed on germination medium (GM) plates containing ½ Murashige and Skoog (MS) solid medium (0.8% agar and 1% sucrose), supplemented with Gamborg’s vitamins solution (SigmaAldrich, Germany). After stratification for three days at 4°C in the dark, the plates were vertically placed in the growth chamber under a 16 h: 8 h, light: dark photoperiod, 120 mol m−2 s−1 photosynthetically active radiation (PAR), 23/20°C day/night temperatures, and 60% relative humidity. To simulate salt stress, EV, B2, and C4 seedlings were exposed to different 16 433 The copyright holder for this preprint bioRxiv preprint doi: https://doi.org/10.1101/2024.02.09.579703 ; this version posted February 14, 2024. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license .

Extracted molecule

total RNA

Extraction protocol

Total RNA from frozen root samples was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The RNA concentration, integrity, and quality were determined with a Qubit RNA Broad-Range Assay Kit (Invitrogen, Waltham, MA, USA) and a Qubit 4 benchtop fluorometer (Invitrogen, Waltham, MA, USA). RNA integrity was further assessed with a 2100 Bioanalyzer
mRNA sequencing was performed at the Michigan State University Research Technology Support Facility Genomics Core (https://rtsf. natsci.msu.edu/genomics/). Libraries were prepared using the Illumina Stranded mRNA Library Preparation, Ligation Kit with IDT for Illumina Unique Dual Index adapters following the manufacturer’s recommendations except that half volume reactions were used. Completed libraries were quality checked and quantified using a combination of Qubit dsDNA high sensitivity (HS) and Agilent 4200 Tape Station HS DNA1000 assays. The libraries were pooled in equimolar proportions and the pool quantified using the Invitrogen Collibri Quantification qPCR kit. The pool was combined with other pools of Illumina Stranded RNA libraries prepared by the Genomics Core to make use of a shared S4 lane. This combined pool was likewise quantified using the Invitrogen Collibri Quantification qPCR kit. The combined pool was loaded onto one lane of an Illumina S4 flow cell and sequencing was performed in a 2x150 bp paired end format using a NovaSeq 6000 v1.5, 300 cycle reagent kit. Base calling was done by Illumina Real Time Analysis (RTA) v3.4.4 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

STAR
The paired-end raw sequences obtained from RNA-seq of WT and C4 grown under both normal (0 mM NaCl) and salt-stress (150 mM NaCl) conditions were checked using FastQC v0.12.1, then trimmed using fastp
High quality reads were aligned to Athaliana_447_TAIR10.fa reference genome (phytozome-next.jgi.doe.gov/) using STAR 2.7.11a (
Read coutn calculated
Assembly: Arabidopis TAIR11
Supplementary files format and content: tab-delimited txt file contatins raw counts for each Sample
Supplementary files format and content: tab-delimited txt file contatins raw counts for each Sample

Submission date

Jun 24, 2024

Last update date

Jun 25, 2024

Contact name

Mostafa ABDELWAHED NOURELDEIN ABDELRAHMAN

E-mail(s)

[email protected]

Phone

8068051625

Organization name

TEXAS TECH UNIVERSITY