Sample 19
Protocols: Samples were collected at 1, 3, 5, and 7 days after zinc starvation. At each time point, plants were separated into root and shoot tissues. Roots were rinsed three times with deionised water and briefly dried on paper towels. Whole root and shoot samples were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction.  Triticum aestivum (cv. Paragon) seedlings were hydroponically grown in a controlled environment chamber using a custom-built aerated hydroponic system. Each seedling was cultivated individually in 1 L culture pots. Growth conditions were maintained at 20 °C during the day and 16 °C at night, with a 16-hour photoperiod. Light was provided by fluorescent bulbs at an intensity of 500 μmol m⁻² s⁻¹. Relative humidity was maintained at 70% during the day and 80% at night.  The composition of the nutrient solution was 1.5 mM Ca(NO₃)₂, 5 mM KNO₃, 2 mM NaNO₃, 1 mM MgSO₄, 0.5 mM KH₂PO₄, 25 μM FeNaEDTA, 0.2 μM CuCl₂, 1 μM H₃BO₃, 0.6 μM MnCl₂, 0.1 μM Na₂MoO₄, 5 μM KCl, 8 μM ZnCl₂, including the chelator 75.5 μM HEDTA. For zinc-deficiency treatments, ZnCl₂ was omitted (0 μM). 2.35 mM MES monohydrate was added as buffer, and the pH was adjusted to 5.8 using KOH. The nutrient solution was changed three times a week.  Seedlings were transferred to the hydroponic system 7 days after germination. For the first 3 days, plants received half-strength nutrient solution, followed by full-strength solution. Zinc-starvation treatment began at 14 days after germination, when plants were supplied with nutrient solution lacking ZnCl₂.  Samples were ground to a fine powder using either a pestle and mortar in liquid nitrogen or a freezer mill (SPEX SamplePrep 6870), depending on sample quantity. Total RNA was extracted from approximately 1 g of ground tissue using a modified protocol based on Verwoerd et al. (1989), as described in Evens et al. (2017). To ensure no DNA carryover, DNase treatment was performed using RQ1 RNase-Free DNase (Promega). After DNase treatment, RNA was further purified by phenol/chloroform/isoamyl-alcohol purification.  Prior to RNA sequencing, total RNA was further purified using the GeneJET RNA Cleanup and Concentration Micro Kit (Thermo Scientific), following the manufacturer's protocol. RNA concentration was determined using the Qubit Broad Range Assay (Invitrogen), and purity was assessed via A260/A280 ratio using a NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA integrity was also evaluated by electrophoresis of 500 ng RNA on a 1% agarose TAE gel.  Library preparation, poly-A selection for rRNA removal, quality control, and multiplexing were performed by Novogene UK following their standard RNA-seq workflow. After fragmentation, first-strand cDNA synthesis was carried out using random hexamer primers, followed by second-strand synthesis using dTTP (for non-directional libraries). Subsequently, end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification were performed. Library quality was assessed using Qubit fluorometry and qPCR quantification, and size distribution was evaluated using a fragment analyser.
ENA first public
2026-08-24
INSDC center name
Post-doctoral Researcher
INSDC status
public
Submitter Id
E-MTAB-15529:Sample 19
broker name
ArrayExpress
collection date
not collected
common name
bread wheat
cultivar
Paragon
developmental stage
Tillering
genotype
wild type genotype
geographic location (country and/or sea)
not collected
growth condition
Zn starvation (0μΜ)
isolate
not applicable
organism part
root
replicate
1
sample name
E-MTAB-15529:Sample 19
scientific_name
Triticum aestivum