2024-04-01
ENA first public
ERC000011
ENA-CHECKLIST
University of Groningen
INSDC center name
public
INSDC status
ERS18444726
SRA accession
E-MTAB-13910:Atha_subm1_I
Submitter Id
ArrayExpress
broker name
native plant cell
cell_type
not collected
collection date
thale cress
common name
Protocols: young leaves including shoot apical meristems were harvested and immediately snap frozen in liquid nitrogen All plant species were grown on a soil mixture until the 10-leaf-stage, about 3-4 weeks after germination, according to established protocols (Müller et al. 2019), under short-day conditions (8 h light, 16 h darkness), 100 µmol photons * m-2 * s-1 and 23 °C. Submergence stress was applied to the plants by immersing them in big, transparent boxes (about 50 l volume) filled with 40 l of tap water 24 h before start of the treatment. Treatment was started 2 hours after start of illumination. Frozen samples were ground for 3 min in 2 ml tubes with zirconia/silica beads using a Mini-Beadbeater (BioSpec Products, Bartlesville, OK, USA) in plates frozen with liquid nitrogen. After grinding, 1 ml lysis binding buffer (Dynabeads mRNA Direct Kit; Thermo Fisher Scientific, Waltham, MA, USA) was added and samples were homogenized with this buffer for an additional 2 min in the Beadbeater. Homogenized samples were centrifuged at 12 000 g for 3 min and loaded onto a homogenizer spin column (Omega Bio-Tek, Norcross, GA, USA) and centrifuged to remove excess tissue. Lysates were transferred to a 96-well plate and stored at −80°C until library preparations. RNA-seq libraries were constructed using breath adapter directional sequencing (BrAD-seq) protocol for non-strand-specific libraries (Townsley et al., 2015). Briefly, 200 μl of lysates was used for messenger RNA (mRNA) isolation, following the manufacturer's instructions (Dynabeads mRNA Direct Kit; Thermo Fisher Scientific). Isolated mRNA was fragmented by reverse transcription (RT) buffer for 90 s at 94°C (RevertAid RT enzyme; Thermo Fisher Scientific), and complementary DNA (cDNA) synthesis was done on the fragmented mRNA. Second-strand synthesis was done according to BrAD-seq protocol and was followed by adapter ligation. PCR enrichment was performed using unique indexed oligos for multiplexing.
description
sporophyte vegetative stage
developmental stage
normal
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Arabidopsis thaliana
organism
Arabidopsis thaliana
organism
shoot
organism part
Arabidopsis thaliana
scientific_name
submerged
stimulus
Atha_subm1_I
title