2025-07-31
ENA first public
ERC000011
ENA-CHECKLIST
Institute of Biology, Biotechnology and Environmental Protection, Faculty of Natural Sciences, University of Silesia in Katowice
INSDC center name
public
INSDC status
ERS23281153
SRA accession
E-MTAB-14835:Sebastian-CTRL
Submitter Id
1 Day After Imbibition
day
age
ArrayExpress
broker name
not collected
collection date
domesticated barley
common name
Sebastian
cultivar
Protocols: We followed the protocol described in Peirats-Llobet et al. (2023). At 1 DAI (days after imbibition), embryos were isolated from the germinating seeds using a scalpel, and the embryonic root was removed. The embryos were embedded in an optimal cutting temperature (OCT) medium and frozen in an isopentane bath on dry ice. The frozen samples were stored at –80°C until further processing. Sections of 10 µm thickness were prepared using a cryostat (Leica CM3050 S) at –18°C. Two control embryo sections and three ABA embryo sections were placed on Visium Spatial Gene Expression Slide. Slides were fixed in chilled methanol for 30 min in –20°C. After fixation, sections were stained for 5 min with 0.1% Safranin O (Sigma-Aldrich, cat. no. S8884-25G) in 50% ethanol. The sections were then washed in an alcohol series (50%, 70%, 100%) for 1 min . Slides were imaged in the bright field using a light microscope (Leica DS5500) for downstream spatial transcriptomic analysis. Seeds were sterilized in a 20% sodium hypochlorite solution for 20 minutes and then thoroughly washed three times with sterile water for 5 minutes. After sterilization, seeds were placed in Petri dishes (⌀ = 90 mm) on three layers of Whatman filters with 5 ml of distilled water (control) or a distilled water containing 75 µM ABA solution (cis–trans-abscisic acid; Sigma-Aldrich, catalog no. 862169). The seeds were stratified in darkness at 4°C for 4 days and then transferred to a growth chamber  (22°C, 16-h-light/8-h darkness, 200 μmol/m−2 s−1 illumination).  RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. RNA integrity was assessed using an Agilent 2100 Bioanalyzer to determine the RNA Integrity Number (RIN).  cDNA libraries were prepared using the Visium Spatial Gene Expression protocol (10x Genomics). Poly-A mRNA was captured and reverse-transcribed to generate cDNA, which was subsequently amplified and indexed for sequencing.
description
Seed germination
developmental stage
wild-type Sebastian
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Hordeum vulgare subsp. vulgare
organism
Hordeum vulgare subsp. vulgare
organism
embryo
organism part
Hordeum vulgare subsp. vulgare
scientific_name
Sebastian-CTRL
title
control
treatment