Brassica napus
Protocols: Developing seeds were sampled at five stages of seed development (stage 1 = pre-storage, stage 2 = early storage, stage 3 = mid storage, stage 4 = late storage, stage 5 = maturation). From stage 2 onwards, seeds were manually dissected into four separate organs/tissues (SC = seed coat, IC = inner cotyledon, OC = outer cotyledon, and RA = radicle). Three biological replicates per stage x organ/tissue combination were generated, comprising pools of dissected seed material from multiple individual siliques and different plants. Depending on the developmental stage (seed size), between two hundred  and five hundred individual seeds were sampled, dissected, and pooled per biological replicate to provide sufficient material (a minimum of approx. 100 mg) for subsequent molecular omics analyses. All samples were snap frozen in liquid nitrogen directly after dissection of the respective seed and stored at -80 °C until RNA extraction. Plants were grown in the container-based system of the PhenoSphere at the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) under controlled environmental conditions in 2020-2021. In each container 48 plants were grown (sowing density: 48 plants/m2). Containers were filled with 1 m3 soil, divided into layers simulating natural soil layers: top soil from the field, loam, coarse sand, and coarse gravel. The climate regime was based on twelve years of hourly records (between 2008 and 2019) from IPK's weather station, and designed to simulate a representative growing season with a dynamic field-like environment, including the simulation of wind and diurnal changes. Plants were watered by an automatic drip irrigation system. During the winter months (November to March), containers were moved outside the facility for hibernation, providing natural cold (negative) temperatures. Total RNA was extracted from sample (5-10 mg aliquot of pooled seed tissue) using the Absolutely RNA Microprep Kit (Agilent Technologies, Santa Clara, California, U.S., Cat #400805) according to the manufacturer's protocol with minor modifications: lysis buffer volume was scaled up to 200 µl per sample and supplemented with 1.5% polyvinylpolypyrrolidone 25 (PVP particle size 25000, Serva, Heidelberg) to remove polysaccharides, proteins, and polyphenol compounds. Sample material was homogenised in the lysis buffer by orbital shaking, incubated for 20 minutes at 50°C and subsequently centrifuged at 10,000g for 2 min to separate the cell debris and PVP from the extract. The supernatant was transferred to a new 1.5 ml Eppendorf tube, mixed with an equal volume of 70% ethanol and immediately transferred to an RNA binding column. During further processing, DNA was removed by on-column DNase I treatment after the first washing-step, following the manufacturer's instructions. Purified RNA was eluted in 20 µl nuclease-free water.  RNA quantity and purity were assessed using a NanoDrop spectrophotometer and QubitTm 4 fluorometer measurements. Subsequently, RNA was diluted to a concentration of 50 ng/µl and 30 µl (total amount of approx. 1500 ng) were provided to the IPK-Gatersleben NGS sequencing lab. Paired-end libraries with a read length of 109 bp were constructed using the Illumina Stranded RNA Prep Kit (Illumina, Berlin, Germany, Product No. 20040534).
ENA first public
2024-01-01
INSDC center name
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Department Molecular Genetics
INSDC status
public
Submitter Id
E-MTAB-14603:L401HPKH21P03S4OC
biosamples identifier
SAMEA115475520
broker name
ArrayExpress
collection date
not collected
common name
rape
cultivar
Express 617
developmental stage
stage 4 (late storage)
genotype
wild type genotype
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
outer cotyledon
sample name
E-MTAB-14603:L401HPKH21P03S4OC
scientific_name
Brassica napus